KR100568664B1 - Cachexia prevention and / or treatment - Google Patents

Abstract

The present invention provides an agent for preventing and / or treating cachexia comprising Tumor Cytotoxic Factor-II (TCF-II) as an active ingredient. The agent of the present invention is based on factors such as cancer, AIDS, heart disease, infectious disease, shock, laceration, endotoxinemia, prosthesis, surgery, diabetes, collagen, radiotherapy, or chemotherapy. Excellent prophylactic and / or therapeutic agents for one cachexia are provided and are useful as a medicament.

Description

Cachexia prevention and / or treatment

The present invention relates to a cachexia prevention and / or treatment agent comprising Tumor Cytotoxic Factor-II (TCF-II) as an active ingredient. According to the present invention, at least one selected from the group consisting of cancer, AIDS, heart disease, infection, shock, laceration, endotoxinemia, proneitis, surgery, diabetes, collagen, radiotherapy, or chemotherapy An excellent prophylactic and / or therapeutic agent for cachexia developed on the basis of factors is provided and is useful as a medicament.

In general, in diseases including cancer, AIDS, and heart disease, anorexia, weight loss, stamina, weakness, skin atrophy or dryness, anemia, swelling, and blood clotting abnormalities are accompanied. The condition of is called cachexia. By falling into this systemic symptom, the patient eventually dies (Tamakumasago City et al .: Ayumi of Medicine, 149, 371-373 (1989)). Furthermore, if active radiation therapy or chemotherapy is actively performed on advanced or terminal cancers that cannot be cured by surgery, bioprotective functions such as immunity are extremely lowered and life is saved against the peculiar nutritional decline as described above. On the contrary, a shortening situation occurs, which is a major problem in treatment. Cachexia has been described as an imbalance in cashiers due to decreased nutritional intake and increased nutrient consumption of diseased organisms, and effects on the metabolism of humoral factors mobilized from cancer tissues or lesions. come. For this reason, active nutritional administration such as high-calorie sap is performed to improve cachexia in order to compensate for overwhelming nutrition or lack of energy and to increase immunity. However, in the state of cachexia, the administered energy is difficult to be used for the purpose of maintaining the patient's life, and in particular, in the case of cholecarcinoma, it becomes a nutrient for cancer cells and results in assisting its proliferation. Could not.

In recent years, monokines and cytokines mobilized from macrophages, such as Tumor Necrosis Factor (TNF), have attracted attention as cachexia adults. TNF has been found as a factor that interferes with cancer cells, and it is evident that macrophages and the like, which have an empty phagocytosis, are secreted by one of the immune cells. Originally, it has a direct killer cell effect and was expected as an anticancer agent from having a strong antitumor activity, but in recent years, it causes weakness such as weight loss of cancer patients and severe infectious patients, namely cachexia, Or it is elucidated to be a cytokine of the pneumothorax causing an inflammatory response, and various actions of TNF have been studied thereafter. The main action of TNF is (1) osteoclast, (2) hyperlipidemia by complicated inhibition of lipids into cells, (3) induction of production of interleukin 1 or colony qualification factor, (4) vascular endothelial disorder, (5) Mediation reaction of exotoxin shock caused by severe infection. By suppressing the increase of TNF from these things, treatment of various diseases, namely, cachexia accompanied with cancer, AIDS, heart disease, infection, shock, laceration, endotoxinemia, organitis, or these diseases Although it is expected to develop drugs for the treatment of various inflammatory diseases including the treatment of chronic arthritis, chronic arthritis, and inflammatory diseases, it is still not obtained satisfactory.

(Initiation of invention)

The present inventors intensively searched for a therapeutic drug for cachexia and found that TCF-II, known as a tumor cell disorder factor, has an excellent preventive and therapeutic effect on cachexia. Therefore, the present invention is based on factors such as cancer, acquired immunodeficiency syndrome (AIDS), heart disease, infectious disease, shock, laceration, endotoxinemia, organitis, surgery, radiotherapy, chemotherapy, etc., which have TCF-II as an active ingredient. The object of this invention is to provide a preventive and / or therapeutic agent for cachexia developed.

The present invention relates to a cachexia prevention and / or treatment agent comprising TCF-II as an active ingredient. According to the present invention, at least one selected from the group consisting of cancer, AIDS, heart disease, infection, shock, laceration, endotoxinemia, proneitis, surgery, diabetes, collagen, radiotherapy, or chemotherapy An excellent prophylactic and / or therapeutic agent for cachexia developed on the basis of factors is provided and is useful as a medicament.

In particular, it is useful for the prevention and / or treatment of cachexia developed on the basis of cancer factors.

Figure 1 shows the improvement effect on the weight loss of TCF-II KYN-2 cell transplanted mice in Example 1. In the figure, ++ indicates a significant difference of 1% or less of risk (P <0.01), and ** indicates a significant difference of 1% or less of risk (P <0.01) after group I administration.

Fig. 2 shows the improvement effect on the hematocrit reduction of KYN-3 cell transplanted mice of TCF-II in Example 1. In the figure, ++ indicates that there is a significant difference of 1% or less in risk (P <0.01).

Fig. 3 shows the inhibitory effect on TNF level increase in ascites of KCF-3 cell transplanted mice of TCF-II in Example 1; In the figure, * indicates a significant difference with respect to group I at 5% or less (P <0.05), and ** indicates a significant difference with respect to group I at 1% or less (P <0.01).

(The best form to carry out invention)

TCF-II, which is an active ingredient of the present invention, is a known protein derived from human fibroblasts, and has the following characteristics.

i) molecular weight (SDS electrophoresis)

   Non-reducing: 78,000 ± 2,000 or 74,000 ± 2,000

     Under reduction: 52,000 ± 2,000 (common band A)

              30,000 ± 2,000 (Band B)

              26,000 ± 2,000 (Band C)

ii) Isoelectric point: 7.4-8.6

The TCF-II is a method of concentrating a human fibroblast culture medium and adsorbing it to an ion exchanger, and purifying the eluate using affinity chromatography (WO90 / 10651) or a genetic engineering method (WO92 / 01053). Obtained by

TCF-II, which is an active ingredient of the present invention, can be derived from fibroblasts, and is produced by microorganisms or other cells by gene replacement operation based on the gene array described in WO90 / 10651. You can do it. Moreover, you may use what was obtained by the genetic engineering method disclosed in WO92 / 01053. At this time, other sugar chains due to differences in host cells or microorganisms, or those which do not bind sugar chains can be used. Preferably, the sugar chains are bound. TCF-II obtained by these methods can be further concentrated and refine | purified by the normal isolation | purification purification method. For example, precipitation with an organic solvent, salting out, gel filtration, affinity chromatography using a monoclonal antibody, electrophoresis and the like can be given. Purification by affinity chromatography using a monoclonal antibody can be purified using the monoclonal antibody disclosed in Japanese Patent Laid-Open No. 5-97. The obtained purified TCF-II can be lyophilized or cryopreserved. In addition, as long as it has the same activity as TCF-II, it can be used as a medicament like the present invention. For example, hepatocyte proliferation factor (HGF; Japanese Patent Application Laid-Open No. 63-22526), which is a protein having a difference between TCF-II protein and 5-amino acid, or purified Scatter Factor [SF; Gherardi and Stocker, Nature, 346, 228 (1990)].

The cachexia prophylactic and / or therapeutic agent of the present invention can be administered as an injection from intravenous, intramuscular or subcutaneous. These formulations are manufactured according to well-known pharmaceutical preparation methods, and a pH adjuster, a buffer, a stabilizer, etc. can be added as needed. When the agent of the present invention is administered to a patient, it varies depending on conditions of the patient's symptoms, health condition, age, weight, etc., and is not particularly limited. Preferably, the formulation containing 6 mg to 60 mg may be administered once or more per day.

Next, although this invention is demonstrated with a manufacture example and an Example, these are only illustrations and this invention is not limited at all by these.

Preparation Example 1

Purification of TCF-II

Cells were cultured according to the method disclosed in WO90 / 10651 and Higashio's method [Higashio, K. et al, BBRC, Vol. 170, pp 397-404 (1990)] to obtain purified TCF-II. That is, 3 × 10 ⁶ human fibroblasts IMR-90 (ATCC CCL-186) were placed in a roller bottle containing 100 ml of DMEM medium containing 5% calf serum, and rotated at a rotational speed of 0.5 to 2 rotations / minute. Incubation was continued for 7 days. When the total cell number became 1 × 10 ⁷ cells, the cells were detached by trypsin, the cells were collected at the bottom of the bottle, sterilized and put into 100 g of 5-9 mesh ceramics (Toshiba Ceramic Co., Ltd.), and the cells were left standing for 24 hours and cultured. Thereafter, 500 ml of the culture solution was added, and the culture was continued. Every 7-10 days, the whole amount of medium was collect | recovered, and fresh medium was supplemented. In this way, production was continued for 2 months, and 4L of culture solution was collected per roller bottle. The specific activity per the obtained culture solution was 32 µg / ml. 750 L of the culture solution was concentrated by UF treatment with a membrane filter (MW 6000 cut; Amicon), and CM- Sephadex C-50 (Palciacia), Con-A Sepharose (Palmacia), and Mono S column (Palma) Four steps of chromatographic purification with Shia Corporation) and heparin-Sepharose (Palmasia Corporation) were carried out to obtain purified TCF-II.

Preparation Example 2

Production of Transgenic TCF-II

According to the method disclosed in WO92 / 01053, cells incorporating the TCF-II gene were cultured to obtain purified TCF-II. Transformed Namalwa cells were cultured to obtain 20 L of culture medium. The culture solution was treated in the order of CM-Sephadex C-50 chromato (Palciacia), Con-A Sepharose CL-6B chromatography (Palciacia) and Mono S column (Palciacia) in the order of HPLC. , About 11 mg of purified TCF-II was obtained.

Preparation Example 3

Preparation of TCF-II Formulations

The manufacture example of the injection of TCF-II obtained by Examples 1 and 2 is shown.

(1) 20 µg TCF-II

    Human Serum Albumin 100mg

The composition was dissolved in citric acid buffer solution at pH 6.03, and the total amount was prepared to 20 ml. After sterilization, 2 ml aliquots were dispensed into a vial bottle, and then lyophilized and sealed.

(2) 40 µg TCF-II

    Tween 80 1mg

    Human Serum Albumin 100mg

The composition was dissolved in physiological saline for injection, and the whole amount was prepared to 20 ml. After sterilization, 2 ml aliquots were added to the vial bottle and sealed after lyophilization.

(3) 20 µg TCF-II

    Tween 80 2mg

    Sorbitol 4g

The composition was dissolved in citric acid buffer solution at pH 6.03, and the total amount was prepared to 20 ml. After sterilization, 2 ml aliquots were dispensed into a vial bottle, and then lyophilized and sealed.

(4) 40 µg TCF-II

    Tween 80 1mg

    2g glycine

The composition was dissolved in physiological saline for injection, and the whole amount was prepared to 20 ml. After sterilization, 2 ml aliquots were added to the vial bottle and sealed after lyophilization.

(5) 40 µg TCF-II

    Tween 80 1mg

    Sorbitol 2g

    1g glycine

The composition was dissolved in physiological saline for injection, and the whole amount was prepared to 20 ml. After sterilization, 2 ml aliquots were added to the vial bottle and sealed after lyophilization.

(6) 20 µg TCF-II

    Sorbitol 4g

    Human Serum Albumin 50mg

The composition was dissolved in citric acid buffer solution of pH 6.03, and the whole amount was prepared in 20 ml. After sterilization, 2 ml aliquots were dispensed into a vial bottle and sealed after lyophilization.

(7) 40 μg of TCF-II

    2g glycine

    Human Serum Albumin 50mg

The composition was dissolved in physiological saline for injection, and the whole amount was prepared to 20 ml. After sterilization, 2 ml aliquots were added to the vial bottle and sealed after lyophilization.

(8) 40 µg TCF-II

    Human Serum Albumin 50mg

The composition was dissolved in citric acid buffer solution at pH 6.03, and the total amount was prepared to 20 ml. After sterilization, 2 ml aliquots were dispensed into a vial bottle, and then lyophilized and sealed.

Example 1

Effect of TCF-II Administration on Cachexia of Human Hepatocellular Carcinoma Transplanted Gall Mouse

The transplanted human hepatocellular carcinoma used KYN-2 and KYN-3 strains in which cell proliferation or cell dispersion was observed by TCF-II in a preliminary experiment in vitro. Cell lines of any, 100 U / ml penicillin, 100 µg / ml streptomycin (Gibcosa), 12 mmol / L sodium bicarbonate, 20% heat-inactivated calf serum (Whittaker Bioproducts) The culture was added and cultured under static conditions at 36 ° C., 5% CO ₂ and 100% humidity. Trypsin-EDTA was added to these cell lines, respectively, and the cells were separated. After washing twice with phosphate buffer (PBS), cell suspension was prepared so as to be 2.0 × 10 ⁷ cells / ml.

The skin of the transplanted portion of 4-5 week old female SCID mice was disinfected with body hair and 70% ethanol, followed by ether anesthesia, followed by transplantation of the prepared tumor cells using a 23G needle. KYN-2 weeks (1.0 × 10 ⁷ dogs / horse) were implanted subcutaneously, and KYN-3 weeks (1.0 × 10 ⁷ dogs / horse) were implanted cells intraperitoneally. Regarding the KYN-2 week subcutaneous transplantation, these transplanted mice were divided into 4 groups each at 3 weeks after transplantation having a diameter of 5 mm and at 5 weeks after transplantation in a KYN-3 week intraperitoneal transplant. The control group administered with solvent alone was group I, 0.3 mg / kg / day TCF-II group, group II, 3.0 mg / kg / day TCF-II group, group III, 30 mg / kg / day TCF-II group, IV group. It was made. TCF-II was administered twice a day twice a day intraperitoneally in KYN-2 week transplanted mice and subcutaneously in KYN-3 week transplanted mice.

In the experiment of KYN-2 week subcutaneous tumor mice, the weight before administration of TCF-II and the weight at the end of administration were measured. In the experiment using KYN-3, before and after administration of TCF-II, blood was collected using heparinized capillary tube (derumosa), which was heparinized from the fundus artery under ether anesthesia, and immediately measured after centrifugation by conventional method. It was. At the end of TCF-II administration, dissection was performed under ether anesthesia, and the ascites TNF value was measured using a Factor-Test-XTM Mouse TNF ELISA Kit (Genzyme). Absorbance measurement was performed using Easy Reader EAR 400 (SLT Laboratories).

The weight change of the solvent and the TCF-II administration group is shown in FIG. 1. In the solvent administration group (Group I), the body weight was markedly decreased to about 20% in two weeks. On the other hand, in the TCF-II administration group (Groups II to IV), weight loss is suppressed depending on the dose, and especially in the Group IV, no significant difference was observed before and after administration, and the body weight after administration was the weight after administration of Group I The value is clearly high compared to. Next, the change of hematocrit of the solvent and TCF-II administration group is shown in FIG. By administration of TCF-II, it was recognized that hematocrit levels in the pale cancer mice were improved, and the progression of anemia accompanying cancer proliferation was suppressed. Moreover, the synergistic inhibitory effect of TCF-II is shown in FIG. TNF, which causes cachexia of cholecarcinoma, showed a significant decrease in group II of 0.3 mg / kg / day, which was dependent on the dose of TCF-II and was the lowest dose. That is, administration of TCF-II markedly improved the state of cachexia, such as weight loss due to cancer proliferation, progression of anemia, and elevation of TNF.

(Industrial availability)

As understood from the above, according to the present invention, cancer, AIDS, heart disease, infection, shock, laceration, endotoxinemia, organitis, surgery, diabetes, collagen disease, radiotherapy, or chemotherapy An excellent prophylactic and / or therapeutic agent for cachexia developed on the basis of one or more factors selected from the group consisting of is provided and is useful as a medicament.

Claims (3)

A composition comprising TCF-Π for preventing and / or treating weight loss by proliferation of cancer cells. A composition comprising TCF-Π for preventing and / or treating anemia due to proliferation of cancer cells. A composition comprising TCF-Π for preventing and / or treating elevation of TNF by proliferation of cancer cells.