JPH0361655B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a gastrointestinal ulcer therapeutic and preventive agent containing an alkylated Fc fragment of human IgG (hereinafter simply referred to as alkylated Fc fragment) as a main component. Until now, the pharmacological effects and medicinal uses of alkylated Fc fragments were unknown. Therefore, it was never used as a medicine. The present inventors have now completed the present invention by discovering that alkylated Fc fragments can be used as a therapeutic and preventive agent for gastrointestinal ulcers. The Fc fragment of human IgG has already been reported as a known component fragment of IgG, for example, as reported by Porter et al. (Biochem. J., 73, 119 (1959)). Molecular weight 45,000 ~ obtained by decomposing original IgG with papain or plasmin
50,000 polypeptide chains, and the recovery method was established by Porter et al. The alkylated Fc fragment specified as an active ingredient in the present invention is obtained by cleaving the disulfide bond of a human IgG Fc fragment and subjecting it to alkylation treatment. An outline of a typical method for recovering alkylated Fc fragments according to the present invention is as follows. PH of a solution containing IgG (protein concentration 2-10%)
6-9, add plasmin or papain, and treat at 20-40°C for 10-30 hours. Next, insoluble matter is removed from this treated solution, and undigested IgG and digestion products are separated by gel filtration treatment. Digestion products are chromatographed using ion exchangers (CM-cellulose and DEAE-cellulose), and human IgG Fc fragments are selectively adsorbed, eluted, and recovered. After collecting the human IgG Fc fragment, it is treated with a reducing agent to cleave disulfide bonds, and then alkylated to obtain an alkylated Fc fragment. As a reducing agent, 2-mercaptoethanol (final concentration 0.75-5.25M), dithiothreitol, or dithioerythritol (final concentration 0.01-0.068M)
etc. are used. Alkylation of Fc fragments is achieved by blocking the SH groups according to conventional methods (Biochemistry, 7(5), 1950-1958 (1968)). Examples of such groups include the following. Lower alkyl amount: methyl, ethyl, n-propyl, etc. N,N-dilower 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: carbamoylmethyl, etc. These blocked products can be produced by a method known per se or a method analogous thereto. Next, we will show experiments conducted to confirm the pharmacological action and effects, clinical trials, acute toxicity tests, dosage, administration method, etc. of the alkylated Fc fragment of the present invention. (1) Pharmacological action and effect The anti-ulcer effect of the alkylated Fc fragment was investigated by causing (a) pyloric ligation ulcer and (b) phenylbutazone ulcer in experimental animals. (a) The method of Shiyai et al. (Gastroenterology, 5,
43, (1945)], a pylorus ligation ulcer was created. Namely, Wistar male rats (body weight
After fasting for 24 hours (200 to 250 g), the stomach was removed under ether anesthesia, and the ulcer that developed in the forestomach was removed using the method of Narumi et al. (J. Takeda Res. Lab., 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 intravenously administered twice, immediately after ligation and 8 hours later. (b) 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 alkylated Fc fragment obtained in Reference Example 1 in the amount shown in Table 2 was administered intravenously, and 30 minutes later, phenylbutazone (suspended in 5% gum arabic) was administered.
200 mg/Kg 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. Score1: Long axis of ulcer 1mm Score2: 1-2mm Score3: 2-3mm Score4: 3-4mm Score5: 4-5mm Score10: >5mm Score25: Perforation The results of (a) and (b) are shown in Table 1 and shown in Table 2. According to Table 1, the effect of alkylated Fc fragments on pylorus ligation ulcers is that when 10 mg/Kg was administered twice, the ulcer incidence was approximately 74% compared to the control when saline was administered.
suppressed. Even at a lower dose of 5 mg/Kg twice, 50% inhibitory activity was observed.

【table】

[Table] According to Table 2, the preventive effect of alkylated Fc fragments on phenylbutazone ulcers is
A significant inhibitory activity of approximately 73% was observed at a dose of 10 mg/Kg of Fc fragment.

[Table] (2) Pharmacological action The antiulcer action mechanism of alkylated Fc fragments was investigated. (a) The inhibitory effect of alkylated Fc fragments 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. The total acidity is 1/50N using phenolphthalein as an indicator.
The total pepsin activity was determined by titration with NaOH, and the total pepsin activity was determined according to the Anson method [Brit.j.Pharmacol., 13, 54. (1958)] using casein as a substrate. The specimen (Fc fragment obtained in Reference Example) was dissolved in sterile physiological saline and administered into the tail vein immediately after ligation. The results are shown in Table 3. When 10 mg/Kg of alkylated Fc fragment was administered to the gastric fluid volume of the control group,
It was suppressed by about 70%, and it was also significantly suppressed at 5 mg/Kg. This tendency was also observed to be similarly suppressed in total acidity and total pepsin activity.

【table】

[Table] () Dosage and method of administration Based on the results of the above test, it is preferable to administer the alkylated Fc fragment at 1 to 100 mg/Kg per day for adults. 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 produced, for example, according to methods known to those skilled in the art as described in the Japanese Pharmacopoeia. The agent for treating and preventing gastrointestinal ulcers containing an alkylated Fc fragment as a main component of the present invention has extremely low toxicity and exhibits remarkable pharmacological effects, making it extremely effective as a pharmaceutical agent for treating and preventing ulcers. Next, reference examples and examples of the present invention will be shown. Reference example 1 (Digestion with plasmin) Add sodium azide to a 3% solution (60 ml) of IgG.
Add 60 mg and adjust the PH to 7.5 using 1N NaOH solution. Add plasmin to a final concentration of 4 cu/ml, and perform digestion at 35°C for approximately 15 hours. After treatment, the pH was adjusted to 6.5, and the mixture was allowed to stand at 4°C for 1 hour, and then centrifuged to remove insoluble matter. Transfer plasmin digestive fluid (approximately 60ml) to Sephadex G-200.
column and undergo gel filtration to separate undigested globulin (7S) and digestion products (Fab+Fc). This digested product was then contacted with a CM-cellulose column (PH7.0) to
Adsorb Fc fragment. After washing with the column,
Develop with a solvent containing 0.01M phosphate buffer (PH7.0) and 0.3M NaCl, and collect Fab and Fc fragments separately. The obtained Fc fragment 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.
Disulfide bonds were broken. Then 0.75~
After adding 5.25M iodoacetic acid and reacting for 1 hour while keeping the pH at 8.0, excess sample was removed using a Sephadex G-25 column. Next, the product was dialyzed against physiological saline, and after sterilization filtration, it was made into a freeze-dried product. Reference example 2 (digestion with papain) 2.5% solution of IgG (20ml; 0.02M EDTA-
5 mg of papain was added to 0.05M phosphate buffer (PH7.5), digested at 37°C for 10-20 minutes, and then cooled with ice water. After cooling, centrifuge to remove insoluble matter, and remove the supernatant.
Fractionation was performed using a Sephadex G-150 column to obtain a 3.5S fraction. This was treated with dithiothreitol at a final concentration of 0.014M at pH 7.5-8.0 for 2 hours at room temperature, and then iodoacetamide was added to a final concentration of 0.2M.
The reaction was allowed to proceed for 1 hour under ice cooling. This is 0.005M of PH8
Dialysis was performed against Tris-HCl and the resulting crystals were centrifuged. The supernatant is the crude alkylated Fc fragment;
The precipitated fraction was collected as alkylated Fc fragments. Example 1 (Oral preparation) (1) Carbamoylmethylated Fc fragment 5.0mg (2) Fine particles for direct injection No. 209 (Fuji Chemical) 46.6mg (3) Crystalline cellulose 24.0mg (4) CM-cellulose 4.0mg ( 5) Magnesium stearate 0.4 mg This mixed powder was compressed into tablets each weighing 80 mg. Example 2 (Intravenous injection) (1) Carboxymethylated Fc fragment 50mg (2) Glucose 100mg (3) Physiological saline 10ml After filtering the above mixture with a membrane filter, sterilization filtration was performed again, and the filtration The liquid was aseptically dispensed into vials, filled with nitrogen gas, and sealed to give an intravenous injection. Example 3 (Capsules) (1) Carbamoylmethylated Fc fragment 50g (2) Lactose 935g (3) Magnesium stearate 15g The above components were mixed uniformly, and 200mg of the mixed powder was filled into hard gelatin capsules. Example 4 (Liposomal formulation) A carboxymethylated Fc fragment is dissolved in 0.01M phosphate buffer (PH7.2) containing 0.125M NaCl to a concentration of about 0.5%. On the other hand, 100 mg of egg yolk phospholipids containing 0.5, 10, and 20% (w/w) phosphatidic acid
Each was dissolved in 10 ml of chloroform and a phospholipid film was formed using a rotary evaporator. Add 1 ml of the above alkylated Fc fragment solution to this.
By adding and shaking, closed fat bodies were formed and alkylated Fc fragments were incorporated to obtain a liposome preparation.

Claims (1)

[Scope of Claims] 1. A gastrointestinal ulcer therapeutic and preventive agent containing an alkylated Fc fragment of human IgG as an active ingredient. 2. 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. 3. The agent for treating and preventing gastrointestinal ulcers according to claim 1, which is in a liquid form for intravenous, intramuscular or oral administration.