JPH0361654B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a treatment and prevention agent for gastrointestinal ulcers, which contains a human IgG H chain (hereinafter simply referred to as H chain) as a main component. Until now, the pharmacological action and medicinal uses of IgG H chain were unknown. Therefore, it was never used as a medicine. The present inventors have completed the present invention based on the assumption that the H chain has an anti-gastrointestinal ulcer effect. The heavy 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 H chain specified as an active ingredient in the present invention is a polypeptide chain with a molecular weight of 50,000±1,500 obtained by cleaving the disulfide bond of IgG from human-derived IgG, and the method for recovering it was established by Fleischman et al. has been done. Note that the disulfide bond may be cut not only at an intermolecular bond but also at an intramolecular bond. An outline of a typical H chain recovery method 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 2-3%. Gently pass nitrogen gas through and add 2-mercaptoethanol to a final concentration of 0.75~
Add to 5.25M and leave at room temperature for 1 hour to perform reduction. Then, it was cooled in an ice water bath, and the same amount of 0.75-5.25M 2-mercaptoethanol was added to it.
Add monoiodoacetamide and adjust the pH of the solution to 1 while keeping it at 8.0 by adding trimethylamine etc.
After reacting for about an hour, excess reagent is removed by dialysis against cold saline. In this reaction, free SH groups in the H chain are blocked with -CH ₂ CONH ₂ . Next, H and L chains were dissociated by dialysis against cooled 1M propionic acid, and passed through a Sephadex G-75 column equilibrated with 1M propionic acid, resulting in two separated H and L chains. It is eluted as a peak. After collecting the H chain fraction, it is subjected to desired known treatments that can be provided as pharmaceuticals, such as dialysis, sterilization, heat treatment, and freeze-drying. In addition to 2-mercaptoethanol (final concentration 0.75-5.25M), reagents for cleaving intermolecular and intramolecular disulfide bonds include, for example, dithiothreitol or dithioerythritol (final concentration 0.01-0.068M). is used. Of course, the H chains used in the present invention are not obtained by the above recovery method, but may be H chains produced by other applicable methods. The SH group in the H chain used in the present invention is preferably blocked [Biochemistry,
7(5), 1950-1958 (1968), Method in
Enzymology xxv , 185], this block is preferably a group that is not easily decomposed, such as a group that forms an S--C bond. In addition to the above-mentioned groups, examples of such groups include the following. Lower alkyl group: methyl, ethyl, n-propyl, etc. N,N-di-lower alkylcarbamide-lower alkyl group: N,N-diethyl-carbamidomethyl Lower alkoxycarbonyl-lower alkyl group: ethoxycarbonylmethyl, ethoxycarbonylethyl, etc. Carboxy -Lower alkyl group: Carboxymethyl, carboxyethyl, etc. Cyano-lower alkyl group: Cyanomethyl, etc. β-Amino-lower alkyl group: -
CH ₂ CH ₂ NH ₂ etc. Benzoyl - lower alkyl group: -
CH ₂ COC ₆ H ₅ etc. Carbamoyl - lower alkyl group: -
These blocked substances, such as CH ₂ CONH ₂ , can be produced by known means or similar means. The active ingredient of the gastrointestinal ulcer preventive and therapeutic agent of the present invention is H
As for the chain, it is sufficient to contain H chain as IgG at a molar ratio of 60 to 100%, and L chain may be contained as the remaining 40% molar ratio. Next, the methods and results of experiments conducted to confirm the pharmacological actions and effects of H chains, clinical trials, acute toxicity tests, dosages, administration methods, etc. will be described. () Pharmacological action and effect: (1) pyloric ligation ulcer and (2) phenylbutazone ulcer were induced in real animals, and the anti-ulcer effect of the H chain was investigated. (1) A pylorus ligation ulcer was created according to the method of Shay et al. [Gastroenterology, 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.
After being left in a fasting state for 16 hours, the stomach was removed under ethereal 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)], 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 (4 mm or more in diameter) 5: Perforation The specimen obtained in Reference Example 1 was used,
This 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 H chain obtained in Reference Example 1 in the amount shown in Table 2 was intraperitoneally administered, and 30 minutes later, 200 mg/Kg of phenylbutazone (5% gum arabic suspension) was administered.
was administered orally. After being left without food or water for 5 hours, the stomach was removed under ether anesthesia and fixed in formalin. For ulcers occurring in the glandular stomach region, the following scores were determined and the summed value was expressed as an 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 H chain on pyloric ligation ulcers is that when 50 mg/Kg and 10 mg/Kg were administered twice each, the ulcer incidence was suppressed to 90% compared to the control physiological saline administration. . Further lowered the dose 5
It was suppressed by 71% with two doses of mg/Kg, and even with two doses of 1 mg/Kg.
Approximately 50% inhibitory activity was observed. [Table] According to Table 2, the preventive effect of H chain against phenylbutazone ulcers was 83% significant inhibitory activity at a dose of 5 mg/Kg of H chain. [Table] () Pharmacological action The anti-ulcer action mechanism of H chain was investigated. (1) The inhibitory effect of H chain on gastric juice secretion was investigated. Administration was performed by dissolving the product obtained in Reference Example 1 in physiological saline and administering the solution 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 is determined by using phenolphthalein as an indicator.
It was determined by titration with 1/50N NaOH, and the total pepsin activity was determined by the Anson method using casein as a substrate [Brit.J.Pharmacol., 13 , 54,
(1958)]. The specimen (H chain obtained in Reference Example 1) 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 H chain was administered intraperitoneally to the gastric fluid volume of the control group, 83
%, and it was also significantly inhibited at 1 mg/Kg. This tendency was also observed to be similarly suppressed in total acidity and total pepsin activity. When administered intravenously, strong gastric juice secretion suppressive activity was observed in both 5 mg/Kg and 1 mg/Kg doses depending on the dose. [Table] [Table] () Toxicity The H chain-containing preparation of the present invention has human IgG
Since it is made from human IgG, it is guaranteed to be as safe as human IgG in terms of toxicity. Incidentally, the results of determining the LD ₅₀ values for IgG and the H chain obtained in Reference Example 1 are as shown in Table 4. [Table] () Dosage and method of administration Based on the results of the above test, the H chain is
Preferably, 100 mg/Kg is administered. This drug can be administered in both injection and oral forms. When used as an injection, for example, it is dissolved in distilled water for injection before use. The method of administration is intravenous and intramuscular. When used as an oral preparation, it is administered as a capsule, tablet, powder, liposome preparation, or oral liquid preparation. These are manufactured according to methods well known to those skilled in the art, such as those described in the Japanese Pharmacopoeia. The agent for treating and preventing gastrointestinal ulcers of the present invention, which has an H chain as a main component, has extremely low toxicity and exhibits remarkable pharmacological effects, making it extremely useful as a drug for treating and preventing ulcers. Next, reference examples and examples of the present invention will be described. Reference Example 1 IgG was dissolved in 0.05M Tris-HCl buffer (PH8.2) to a concentration of about 2%, and 2-mercaptoethanol was added to a final concentration of 0.75 to 5.25M to cleave disulfide bonds. Next, 0.75-5.25M iodoacetic acid was added, the pH was maintained at 8.0, and the reaction was carried out for 1 hour.
Excess reagent was removed using a Sephadex G-25 column. Next, it was applied to a Sephadex G200 column (4.0 x 120 cm) [solvent: 0.04M SDS - 0.05M phosphate buffer (PH8.0)] in the presence of SDS (sodium dodecyl sulfate), and measured at OD280 mm, and the H chain fraction was measured. Collected the amount. SDS was removed from the H chain fraction, dialyzed against physiological saline, and after further sterilization, a freeze-dried product was obtained. Reference Example 2 IgG is dissolved in 0.5M Tris-HCl buffer (PH8.2) containing 2mMEDTA to a concentration of approximately 2%. After passing nitrogen gas for about 20 minutes, dithiothreitol was added to a final concentration of 0.01 to 0.068 M, and the mixture was reacted at room temperature for 2 hours. Then add iodoacetamide to the final concentration.
After adding 0.04 to 0.272M and reacting for 1 hour under ice-cooling, dialysis was performed at 4°C against 0.1M acetic acid, buffer solution (PH5.5), and distilled water in this order. Note that all operations up to now were performed under light-shielding conditions. Next, add propionic acid to the solution to a final concentration of 1M, and immediately
Cephadex G equilibrated with 1M propionic acid
Gel filtration was performed on a −100 column. Measurement was carried out at OD280 nm, and the H chain fraction was collected, concentrated by ultrafiltration, dialyzed against distilled water, further sterilized, and freeze-dried. The yields obtained when the above operations were carried out were as shown in Table 5. [Table] Example 1 (Oral preparation) (1) Carbamoylmethylated H chain derived from human IgG 5.0mg (2) Fine particles for direct application No. 209 (manufactured by Fuji Chemical) 46.6mg 〓Magnesium aluminate metasilicate 20% Corn Starch 30% Lactose 50% (3) Crystalline cellulose 24.0mg (4) Carboxymethyl cellulose/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) Carboxymethylated H chain derived from human IgG 30mg Carboxymethylated L chain derived from human IgG 20mg (2) Glucose 100mg (3) Physiological saline 10ml (3) and (1) Add (2) at the above polymerization ratio and stir.
Dissolve completely. After the lysate is sterilized using a membrane filter with a pore size of 0.45 μm, sterilization 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 (Capsule) (1) Carboxymethyl H chain derived from human IgG 50g (2) Lactose 935g (3) Magnesium stearate 15g The above components were each weighed and mixed uniformly to a total of 1000g to form a mixed powder. in hard gelatin capsules
Fill 200mg each. Example 4 (ribosome preparation) Carbamoylmethylated H chain derived from human IgG was
0.01M phosphate buffer (PH7.2) containing 0.125M NaCl
Dissolve to a concentration of approximately 0.5%. On the other hand, 0, 5, 10,
100 mg of egg yolk phospholipids containing 20% (w/w) phosphatidic acid were each dissolved in 10 ml of chloroform, and a phospholipid film was formed using a rotary evaporator. Add the above H chain solution 1 to this
ml was added and shaken to form a closed fat body, and the H chain was incorporated to obtain a liposome preparation. Centrifuge this fat body (10000 rpm, 30 minutes)
The proteins incorporated therein were measured. The results are shown in Table 6. [Table] [Table]

Claims (1)

[Claims] 1. A gastrointestinal ulcer treatment and prevention agent containing a human IgG H chain as an active ingredient. 2. The agent for treating and preventing gastrointestinal ulcers according to claim 1, wherein the H chain is an alkylated H chain. 3. The agent for treating and preventing gastrointestinal ulcers according to claim 1, which is in the form of a powder, tablet, capsule, or liposome preparation 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.