JPH0640934A - Therapeutic agent for hepatopathy comprising tcf-ii as active ingredient - Google Patents

Abstract

PURPOSE:To obtain a therapeutic agent for hepatopathy comprising a glycoprotein tumor cytotoxic factor (TCF)-II derived from a human fibroblast as an active ingredient. CONSTITUTION:The therapeutic agent for hepatopathy comprises TCF-II prepared by concentrating a human fibroblast cultured solution on an ion exchanger and subjecting the resultant eluate to the affinity chromatography or obtainable according to a genetic engineering technique as an active ingredient. This therapeutic agent is administered as a parenteral injection. In this case, the administration is performed by any of intravenous, intraarterial, intramuscular or subcutaneous injection. This therapeutic agent is effective against acute and chronic hepatitis, cirrhosis, fatty liver or biliary congestive hepatopathy.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a therapeutic agent for liver diseases containing TCF-II, a glycoprotein produced by human fibroblasts, as an active ingredient.

[0002]

It is widely known that physiologically active substances produced by human-derived fibroblasts, for example, β-interferon as a tumor cytotoxic factor exist. In addition to this β-interferon, as a physiologically active substance produced by fibroblasts, C described in JP-A-58-146293 is disclosed.
Tumor cytotoxic glycoprotein called BF, JP-A-61-33
Tumor growth inhibitory factor (INF) having a molecular weight of 35,000 to 45,000 described in JP-A No. 120, tumor growth factor FNF described in JP-A No. 61-1872, and molecular weight of 40,000 to 6 described in 62-103021
0,000, a physiologically active substance having a cytotoxic effect with an isoelectric point of pH 5.0 ± 0.5, a molecular weight of 36,000 ± 1,00 obtained from the culture supernatant of human-derived fibroblasts described in JP-A-64-10998.
0, tumor cell damaging factors and the like having a specific amino acid sequence at an isoelectric point of pH 10.5 or higher are known. In the process of studying human fibroblast-derived antitumor protein,
We have discovered a novel antitumor substance that is completely different from the previously reported proteins, succeeded in cloning the cDNA encoding this protein, and confirmed the full amino acid sequence and confirmed its usefulness. This novel antitumor protein and its gene have been filed by the present applicant and have been published as International Publication No. 90/10651. This novel antitumor protein is named TCF-II. This TCF-II has a strong antitumor activity and a growth activity of normal cells, and further, HG which is a growth factor of liver parenchymal cells.
It was confirmed to be a member of the diverse family of F.
TCF-II is 78,0 by molecular weight measurement by SDS electrophoresis.
It has a molecular weight of 00 ± 2,000 or 74,000 ± 2,000, and when reduced, it has a common band of 52,000 ± 2,000 with A chain
Two bands (B chain and C chain) of 0,000 ± 2,000 or 26,000 ± 2,000 are shown. Since TCF-II is a growth factor for liver parenchymal cells, it can be used for the purpose of liver regeneration after hepatectomy, and although such studies have been conducted,
There is no case that its effect was confirmed by animal experiments, and it is not known that it can be used as a therapeutic drug for liver diseases.

[0003]

DISCLOSURE OF THE INVENTION The present inventors have TCF-
Focusing on the physiological activity of II, we have investigated its use as an antitumor agent and as a marker for diagnosis of diseases. The present inventors have found from the process of studying the action of TCF-II on the liver that TCF-II has not only the proliferation of hepatocytes but also a therapeutic effect on various liver diseases. Up to now, it has not been confirmed that TCF-II has a therapeutic effect on various liver diseases, and it was found by the present inventors for the first time that it has a therapeutic effect on various liver diseases. An object of the present invention is to provide a therapeutic agent for liver disease containing TCF-II as an active ingredient.

[0004]

The present invention relates to a liver disease therapeutic agent containing TCF-II as an active ingredient. The active ingredient of the present invention is a known glycoprotein derived from human fibroblasts as described above. This TCF-II has a molecular weight measured by SDS electrophoresis of 78,000 ± 2,000 or 74,0 in the non-reduced state.
00 ± 2,000, and under reduction, 52,000 ± 2,000 common band A, 30,000 ± 2,000 band B and 26,000 ± 2,000
0 band C and two bands. The isoelectric point is
It is a glycoprotein consisting of 723 amino acid sequences from 7.4 to 8.6. The TCF-II can be obtained by concentrating a human fibroblast culture medium, adsorbing it on an ion exchanger, and subjecting the eluate to affinity chromatography (W.
O90 / 10651) or genetic engineering method (WO
92/01053).

That is, TCF-II is the above-mentioned WO90.
It is possible to use human fibroblast-derived cells obtained by the method disclosed in Japanese Patent Publication No. / 10651. 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. TCF-
As a method for producing II, the one produced by the method filed by the present inventors and published as WO92 / 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 obtained purified TCF-II can be freeze-dried or frozen and stored.

The therapeutic agent for liver diseases of the present invention can be administered as an injection. In this case, any of intravenous injection, arterial injection, intramuscular injection or subcutaneous injection may be used. Further, if necessary, it can be used in combination with a drug used for the treatment of liver disorders such as amino acid, vitamin, phospholipid, malotilate, prednisolone, and glycyrrhizin. The dose of TCF-II contained in the therapeutic agent for liver diseases of the present invention is determined by the symptoms of the administration patient, the medical condition, the age, etc., but is preferably 100-30,000 μg as purified TCF-II per adult, preferably, A formulation containing 500-3000 μg can be administered 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 with reference to the following 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, 1990)
The cells were cultured according to the above procedure to obtain purified TCF-II. 5% of human fibroblast IMR-90 (ATCC CCL 186) cells
3 × 10 ⁶ cells were transferred to a roller bottle containing 100 ml of DMEM containing calf serum, and the culture was continued for 7 days while rotating at a rotation speed of 0.5 to 2 rotations / minute. Total number of cells is 1 x 10
^{When the} number of cells reaches ^{7, the} cells are detached with trypsin and collected on the bottom of the bottle.
00 g (manufactured by Toshiba Ceramic Co., Ltd.) was sterilized and charged, and the mixture was left standing for 24 hours and cultured. Then add 500 ml of the above culture,
The culture was continued. The entire amount of the medium was collected every 7 to 10 days, and the medium was replenished with fresh medium. 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 thus obtained was 32 μ /
It was ml. 750 liters of the culture solution was UF concentrated by treatment with a membrane filter manufactured by Amicon (MW 6000 cut), CM Sephadex C-50 (manufactured by Pharmacia),
ConA Sepharose (Pharmacia), Mono
S-column (Pharmacia) Heparin Sepharose (Pharmacia) 5-step chromatographic purification was performed to obtain purified TCF-II having a specific activity of 5248000 u / mg.

[0008]

Example 2 Production of Recombinant TCF-II According to the method disclosed in WO92 / 01053, TCF was prepared.
-Cultured cells containing the -II gene and purified TCF-II
Got The transformed Namalwa cells were cultured to obtain 20 liters of the culture solution. This culture is CM-Sephadex C-50.
Chromatography, Con-A Sepharose CL-6B Chromatography, MonoS
The treatment was carried out in order of HPLC equipped with a column to obtain about 11 mg of active TCF-II.

[0009]

Example 3 Production Example of TCF-II Formulation In this example, a production example of an intravenous, subcutaneous, or intramuscular injection formulation of the recombinant TCF-II obtained by the method of Example 2 above. showed that. (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 amount of 20 ml, and after sterilization, 2 ml each was dispensed into vials, and freeze-dried and sealed.

(3) TCF-II 20 μg Tween 80 2 mg sorbitol 4 g 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. (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 amount of 20 ml, and after sterilization, 2 ml each was dispensed into a vial, which was 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 volume of 20 ml, and after sterilization, dispensed in 2 ml aliquots and freeze-dried. 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 to make a total volume of 20 mg.
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 volume of 20 ml, and after sterilization, dispensed in 2 ml aliquots, freeze-dried and sealed. did. (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.

[0013]

The present invention provides a therapeutic agent for liver diseases containing TCF-II as an active ingredient. The effects of the present invention will be described below with reference to experimental examples in which the effects of the therapeutic agent produced in this example were confirmed.

[0014]

[Experimental Example 1] Therapeutic effect on acute liver injury, chronic liver injury and cirrhosis (1) Method 70% hepatectomy for normal liver, acutely injured liver (hepatitis type, parenchymal cell injury type), chronically injured liver and cirrhosis The therapeutic effect was confirmed. Using male Wistar rats,
7-week-old (body weight 200 g) 70% liver resection, normal liver rat (n = 6), 500 μg / kg galactosamine subcutaneously immediately after hepatectomy, acute hepatitis rat (n = 9-10), liver Immediately after excision
Acute parenchymal cell injury liver rats (n = 10) were orally administered with 0.3 ml / kg of carbon tetrachloride. Also, from 7 weeks of age
Rats given 0.7 ml / kg of carbon tetrachloride orally twice a week for 4 or 10 weeks were subjected to the same liver resection, and chronically injured liver rats (n = 10) and cirrhosis rats (n = 9- 10) were produced respectively. In rats with chronically damaged liver, 0.
Oral administration of 3 ml / kg of carbon tetrachloride caused acute symptoms similar to those in the rat with acute parenchymal cell injury, and the reactivity to TCF-II was compared. TCF-II was intravenously administered at intervals of 12 hours immediately after hepatectomy, and 20 or 100 was given to normal hepatectomy rats.
, 500 μg / kg 3 doses, 500 for other rats
μg / kg was administered and compared with the PBS administration group supplemented with 0.1% human serum albumin. TCF-II for normal hepatectomy rats
To examine the effects of the drug, thrombotest levels and serum triglycerides at 48 hours after surgery, serum total protein and serum HDL cholesterol at 72 hours after surgery, liver weight and liver at 96 hours after surgery.
DNA amount and total liver protein amount were measured. (2) Results Delayed thrombotest levels and decreased serum triglycerides, serum total proteins and HDL cholesterol were observed by 70% hepatectomy, but TCF-II improved all parameters in a dose-dependent manner. Thrombotest values at 48 hours after hepatectomy (FIG. 1), plasma fibrinogen (FIG. 2), serum triglyceride (FIG. 3) and serum total protein (FIG. 4) in normal liver, acutely damaged liver, chronically damaged and cirrhotic rats, respectively. Liver weight (Fig. 5), total liver protein amount (Fig. 6), liver
The amount of DNA (Fig. 7) was the same in the TCF-II administration group in all cases.
These parameters were improved compared with the PBS administration group supplemented with 0.1% human serum albumin.

[0015]

[Experimental Example 2] Effect on fatty liver (1) Method 250 mg of DL-ethionine was added to 7-week-old Wistar male rats.
It was intraperitoneally administered repeatedly at a dose of / kg for 4 days. TCF-II (50 and 500 μg / kg) was added to these rats (n = 10).
Was administered intravenously at 12-hour intervals, and prothrombin time (pT) at 48 hours, antithrombi II activity (AT III), urea nitrogen (BUN), total cholesterol (T-CHO), phospholipid (P
L), HDL cholesterol (HDL) and the number of mitotic cells per 1000 hepatocytes (Mitotic index) and total protein (TP), transaminase (GOT) and lactate dehydrogenase (LDH) in serum at 72 hours Was measured. (2) Results Table 1 shows the average value and standard error value of each measured value. TCF
-II improved the symptoms of ethionine fatty liver rats in a dose-dependent manner at a dose of 50 μg / kg or more.

[0016]

[Table 1]

[0017]

[Experimental Example 3] Effect on cholestatic liver injury (1) Method 50 mg / kg of α-naphthyl isothiocyanate was orally administered to 7-week-old male Wistar rats. TCF-II is α
-Immediately after the administration of naphthyl isothiocyanate, it was administered twice intravenously at 12-hour intervals. Blood was collected 12 hours after TCF-II administration, and transaminases (GOT, GPT) in serum,
Alkaline phosphatase (ALP) P, γ-glutamyl transpeptidase (γ-GTP), total bilirubin (T-BIL)
Direct bilirubin (D-BIL) and liver foreign body excretion function test (BSP test) were performed. (2) Results Table 2 shows the average value and standard error value of each measured value. TCF
-II showed improvement in each parameter at the dose of 500 μg / kg, and improved various symptoms of cholestasis-type liver injury.

[0018]

[Table 2]

[0019]

[Experimental Example 4] Effect on acute hepatitis (1) Method 1 g / kg of galactosamine was subcutaneously administered to 7-week-old male Wistar rats (n = 8). Immediately after the administration of galactosamine, 250 μg / kg of TCF-II was intravenously administered 4 times at 4-hour intervals, and this was repeated for 2 days. After administration of galactosamine
Blood was collected at 48 hours and serum transaminase (G
OT, GPT), γ-glutamyl transpeptidase (γ
-GTP), serum total protein (TP), total cholesterol (T-CHO),
Triglyceride (TG), phospholipid (PL), β-lipoprotein (β
-Lipo), liver weight, liver protein content, and liver DNA content were measured. (2) Results Table 3 shows the average value and standard error value of each measured value. TCF
-II showed improvement in all parameters at the dose of 250 μg / kg, and improved various symptoms of acute hepatitis.

[0020]

[Table 3]

[0021]

[Experimental Example 5] Therapeutic effect of the administration method (1) Method Using Wistar male rats, 70% of the liver was excised at 7 weeks (body weight: 200 g) in a normal liver rat.
The intensity of CF-II was compared between two intravenous administration methods (n = 6) and continuous intravenous drip infusion method (n = 12) at 12-hour intervals. The dose was set to 1 mg / kg / day, serum total protein, albumin, triglyceride, HDL-cholesterolol and thrombotest at 48 hours after hepatectomy were measured, and liver weight was further measured.
The liver regeneration rate was calculated from the excised liver weight. (2) Results Serum total protein (Fig. 8), albumin (Fig. 9), HDL-cholesterol (Fig. 10), triglyceride (Fig. 11), regeneration rate (Fig. 12) by administration of TCF-II after 70% liver resection.
And the changes in the thrombotest values (Fig. 13) are shown. The improvement effect of continuous drip was shown for all parameters. From the above experimental results, the therapeutic agent for liver disease according to the present invention works effectively against liver disease in humans, which has been difficult to treat until now.

[Brief description of drawings]

FIG. 1 shows thrombotest values for rats with various pathological conditions after liver resection.

FIG. 2 shows the amount of serum fibrinogen in rats with various pathological conditions after hepatectomy.

FIG. 3 shows the amount of serum triglyceride in hepatectomized rats with various pathological conditions.

FIG. 4 shows the total serum protein amount in rats with various pathological conditions after hepatectomy.

FIG. 5 shows liver weight of hepatectomized rats with various pathological conditions.

FIG. 6 shows the amount of liver protein in rats undergoing liver resection with various pathological conditions.

FIG. 7 shows the amount of liver DNA in liver-excised various pathological rats.

FIG. 8 shows the total amount of serum protein in intermittently or continuously administered TCF-II to hepatectomized rats.

FIG. 9 shows the amount of serum albumin after intermittent or continuous administration of TCF-II to hepatectomized rats.

FIG. 10 shows the HDL-cholesterol level when TCF-II was intermittently or continuously administered to hepatectomized rats.

FIG. 11 shows serum triglyceride levels when intermittently or continuously administered TCF-II to hepatectomized rats.

FIG. 12 shows the liver regeneration rate when intermittently or continuously administered TCF-II to hepatectomized rats.

FIG. 13 shows thrombotest values when TCF-II was intermittently or continuously administered to hepatectomized rats.

Claims (2)

[Claims] 1. A therapeutic agent for liver diseases, which contains TCF-II as an active ingredient. 2. The therapeutic agent for liver disease according to claim 1, wherein the liver disease is acute hepatitis, chronic hepatitis, cirrhosis, fatty liver, or cholestatic liver disease.