JPS6337089B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a gastrointestinal ulcer therapeutic and preventive agent containing the L chain of human IgG (hereinafter simply referred to as L chain) as a main component. Until now, the pharmacological effects of the IgG L chain were unknown. Therefore, it was never used as a medicine. The light chain of IgG has already been reported as a constituent fragment of known IgG, for example, as reported by Fleischman et al. [Arch.Biochem.
Biophys., Supple) (1), 174, (1962)].
The L chain specified as an active ingredient in the present invention is a polypeptide chain with a molecular weight of 23,000±1,000 obtained from human-derived IgG by cleaving the disulfide bond of IgG, and the method for recovering it was established by Fleischman et al. has been done. An outline of a typical method for recovering L chain according to the present invention is as follows. IgG is dissolved in 0.55M Tris-HCl buffer, pH 8.2, to a concentration of approximately 2%. After gently passing nitrogen gas, 2-mercaptoethanol was added to the solution to a final concentration of 0.75M, and the solution was left at room temperature for 1 hour to perform reduction. Next, it was cooled in an ice water bath, and 2-mercaptoethanol and the same amount of 0.75M monoiodoacetamide were added thereto, and the pH of the solution was maintained at 8.0 by adding trimethylamine, and the reaction was allowed to proceed for 1 hour. The excess was dialyzed against cold saline. Excludes reagents. In this reaction, free SH groups are blocked by alkylation.
Next, H and L chains were dissociated by dialysis against cooled 1M propionic acid, and passed through a column of Sephadex G-75 equilibrated with 1M propionic acid, resulting in two separated H and L chains. Eluted as a peak. After collecting the L chain fraction, it is subjected to desired known treatments such as dialysis, sterilization, heat treatment, freeze drying, etc. so that it can be provided as a pharmaceutical product. Of course, the L chain used in the present invention is not obtained only by the above recovery method, but may be an L chain produced by other applicable methods. The SH group of this L chain may be protected by a pharmacologically acceptable treatment such as alkylation or sulfonation. Next, the methods and results of experiments conducted to confirm the pharmacological action and effect of the L chain, clinical trials, acute toxicity tests, dosage and administration method, etc. will be described. () Pharmacological effects The effects of the L chain were investigated by causing (1) pyloric ligation ulcers and (2) phenylbutazone ulcers in experimental animals. (1) Shay et al.'s method [Gastroentero]
A pylorus ligation ulcer was created according to the method described in Physiology, 5 , 43, (1945)]. Specifically, male Wistar rats (weight 200 to 250 g) were fasted for 24 hours, and then the pylorus was ligated under ether anesthesia. 16 under fasting and water fasting
After leaving the stomach for some time, the stomach was removed under ether anesthesia, and the ulcer that had developed in the forestomach was removed using the method of Narumi et al. [J.Takeda Res.Lab., 29 , 85, (1970)].
Accordingly, the following ulcer index was used for evaluation. 0: Normal 1: Erodion or bleeding spot 2: 10 or less small ulcers (1 mm or less in diameter) 3: 10 or more small ulcers or 10 or less medium ulcers (2-4 mm in diameter) 4: 10 or more medium ulcers Ulcer or large ulcer (diameter 4
mm or more) 5: Perforation The specimen was dissolved in sterile physiological saline, and the amount shown in Table 1 was intraperitoneally administered twice, immediately after ligation and 8 hours later. (2) Suzuki et al.'s method [Japan J.Pharmaco., 26 ,
471 (1976)] to create phenylbutazone ulcers. Wistar male rat (weight 150-200g)
After fasting for 24 hours, the amount of the sample shown in Table 2 was administered intraperitoneally, and 30 minutes later, phenylbutazone (5%
Suspended in gum arabic) 200mg/Kg was orally administered. After being left without food or water for 5 hours, the stomach was removed under ether anesthesia and fixed in formalin. Ulcers that occur in the glandular stomach area are scored as follows:
The summed value was expressed as the ulcer index. Score 1: Long diameter of ulcer 1 mm Score 2: 1 to 2 mm Score 3: 2 to 3 mm Score 4: 3 to 4 mm Score 5: 4 to 5 mm Score 10: > 5 mm Score 25: Perforation The results of (1) and (2) are shown in Table 1 and shown in Table 2. According to Table 1, the effect of L chain on pyloric ligation ulcers is that when L chain was administered twice at 50 mg/Kg and 10 mg/Kg each, the ulcer incidence was suppressed to about 90% compared to the control when saline was administered. be done. Further lowered the dose 5
It was suppressed by 68% with two doses of mg/Kg, and even with two doses of 1 mg/Kg.
Approximately 40% inhibitory activity was observed.

[Table] According to Table 2, the preventive effect of L chain against phenylbutazone ulcers was 82% significant at a dose of 5 mg/Kg of L chain.

[Table] () Pharmacological action The anti-ulcer action mechanism of the L chain was investigated. (1) The inhibitory effect of L chain on gastric juice secretion was investigated. Administration was performed intraperitoneally as well as intravenously. The gastric juice secretion suppressing activity was measured according to the method of Shay et al. [Gastroenterology 26 , 906, (1954)]. That is, the volume, total acidity, and total pepsin activity of retained gastric fluid 4 hours after ligating the pylorus of male Wistar rats (body weight 150 to 200 g) that had been fasted for 48 hours were measured. Total acidity was determined by titration with 1/50N NaOH using phenolphthalein as an indicator.
Total pepsin activity was determined by the Anson method using casein as a substrate [Brit.J.Pharmacol., 13 ,
54, (1958)]. The specimen was dissolved in sterile physiological saline and administered intraperitoneally or intrafemorally immediately after ligation. The results are shown in Table 3. When 5 mg/Kg of L chain was administered intraperitoneally, the amount of gastric juice in the control group was suppressed by 73%, and it was also significantly suppressed at 1 mg/Kg. This tendency was also observed to be similarly suppressed in total acidity and total pepsin activity. When administered intravenously, 5mg/
Strong gastric juice secretion suppressive activity was observed in both Kg and 1 mg/Kg administrations depending on the dose.

[Magnesium metasilicate aluminate 20% Corn starch 30% Lactose 50%]

(3) Crystalline cellulose 24.0mg (4) Carboxylmethylcellulose/calcium
4.0mg (5) Magnesium stearate 0.4mg Pass all of (1), (3) and (4) through a 100 mesh sieve in advance. These (1), (3), (4), and (2) are each dried to reduce the moisture content to a certain level, and then mixed using a mixer in the above weight ratio. Add (5) to the uniformly mixed powder, mix for a short time (30 seconds), and tablet the mixed powder (punch: 6.3mmφ, 6.0mmR) to make one tablet.
It was made into an 80mg tablet. The tablets may be coated with a commonly used gastric soluble film coating agent (eg, polyvinyl acetal diethylamino acetate) or an edible coloring agent, if necessary. Example 2 (Intravenous injection) (1) Carbamoylmethylated L chain derived from human IgG 50 mg (2) Glucose 100 mg (3) Physiological saline 10 ml (3) Add (1) and (2) in the above weight ratio Add and stir;
Dissolve completely. After this solution is filtered using a membrane filter with a pore size of 0.45 μm, sterilization filtration is performed again using a membrane filter with a pore size of 0.20 μm. Aseptically dispense 10 ml of the filtrate into vials, fill with nitrogen gas, and seal to prepare an intravenous injection. Example 3 (Capsules) (1) Carbamoylmethylated L chain derived from human IgG 50g (2) Lactose 935g (3) Magnesium stearate 15g Weigh each of the above components and uniformly mix a total of 1000g to form a mixed powder. into hard gelatin capsules
Fill 200mg each.

Claims (1)

[Claims] 1. A gastrointestinal ulcer therapeutic and preventive agent containing the L chain of human IgG as an active ingredient. 2. The agent for treating and preventing gastrointestinal ulcers according to claim 1, wherein the L chain is an alkylated L chain. 3. The agent for treating and preventing gastrointestinal ulcers according to claim 1, which is in the form of a powder, tablet, or capsule for oral administration. 4. The agent for treating and preventing gastrointestinal ulcers according to claim 1, which is in a liquid form for intravenous, intramuscular or oral administration.