JPS6013717A - Remedy and preventive for ulcer of digestive organ - Google Patents

Abstract

PURPOSE:A remedy and preventive for ulcer of digestive organs, having extremely low toxicity, improved antiulcer actions such as inhibitory activity against secretion of acid in the stomach, inhibitory activity against total pepsins, etc., containing an alkylated Fc fragment of human IgG as an active ingredient. CONSTITUTION:A remedy and preventive for ulcer of digestive organs, containing an alkylated Fc fragment of human IgG. The alkylated Fc fragment is obtained by scossoring disulfide bond of the Fc fragment of human IgG, alkylating it. It is found that the alkylated Fc fragment has low toxicity, improved antiulcer action, and is extremely effective as a remedy and preventive for ulcer. The medicinal action of it has inhibitory activity against secretion of acid in the stomach, inhibitory activity against total acidity and total pepsins. It is used as powder, tablet, capsule, liposome pharmaceutical preparation, etc. for oral medication, or venoclysis, or intramuscular injection. A dose is preferably 1- 100mg/kg/day.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a gastrointestinal ulcer therapeutic and preventive agent containing an alkylated Fc fragment (hereinafter simply referred to as alkylated Fc fragment) of Human 1 (IG) as a main component.

Hitherto, 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 1gG has already been reported as a constituent fragment of known IQG, for example, as reported by Porter et al.
ochem, J., 73.119 (1959))
There is. This person 1 (the l”c fragment of IG is human-derived b+
The molecular weight obtained by decomposing G with papain or plasmin is 45,000 to 50. It is an OOO polypeptide chain,
The recovery method was established by Porter et al.

Alkylated F specified as an active ingredient in the present invention
The c fragment is obtained by cleaving the disulfide bond of the Fc fragment of human 1 (IG) and alkylating it to obtain 1q.

A typical recovery method for alkylated Fc fragments according to the present invention is summarized as follows.

A solution containing IQG (protein concentration 2-10%) was adjusted to pH 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 gG and digestion products are separated by gel filtration. Digestion products include ion exchangers (CM-cellulose and DEAE
- cellulose),
Fc fragments of human IgG are selectively adsorbed, eluted and recovered.

After collecting the human gG Fc fragment, it is treated with a reducing agent to cleave disulfide bonds, and then alkylated to obtain an alkylated Fc fragment.

As the reducing agent, 2-mercaptoethanol, dithiothreitol, dithiothreitol, etc. are used, and the amount used is an amount corresponding to a final concentration of 0.01-0.068M.

Alkylated Fc fragments are created by blocking SH groups according to conventional methods (8io-chemi
stry, 7 (5), 1 950-1 958 (
1968)]. Examples of such groups include the following.

■ Lower alkyl group: methyl, ethyl, n-propyl, etc. ■ N,N-di-lower alkylcarbamide-lower alkyl 4: N, N-diethyl-carbamidomethyl ■ Lower alkoxycarbonyl group: ethoxycarbonylmethyl, ethoxycarbonylethyl, etc. ■ Carboxy-lower alkyl group: carboxymethyl, carboxyethyl, etc. ■ Cyano-lower alkyl group, dianomethyl, etc. ■ β-Amino-lower alkyl group, CH2CH2NH
2 etc.■ Benzoyl-lower alkylation treatment CH2C0C6H
These blocked products such as No. 5 can be manufactured by means known per se or means equivalent thereto.

Next, we will show experiments conducted to confirm certain functions 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 action of the alkylated Fc fragment was investigated by causing (a) pyloric ligation ulcer and (b) phenylbutacin FIAs in experimental animals.

(a> Shai et al.'s method (Gastroen, tero
Logy.

5.43. (1945)), a pylorus ligation ulcer was created. That is, male Wistar rats (body weight 200
After fasting for 241+8 days, the stomach was removed under anesthesia with 1 ether, and the ulcer that had developed on the anterior back was removed using Narumi.
(J, Takeda Res.

Lab, 29, 85. (1970)), the following ulcer index was used for evaluation.

O: Normal 1: Erodion or bleeding spots 2: 10 or less small W4 ulcers (diameter 1# or less) 3: 10 or more small ulcers or 10 or less medium W4 ulcers (diameter 2,4
Marriage) 4: Medium F of 10g or more! I ulcer or large ulcer (diameter of 4 shoes or more) 5: As the perforated specimen, use the one obtained in Reference Example 1, dissolve it in sterile physiological saline, and perform the following procedures in Table 1 immediately after ligation and after 8 hours.
The same drug shown in Figure 1 was administered intravenously twice.

(b) Meki et al.'s method (Japan J, Pharma
co,.

26.471 (1976)) to create phenylbutacin ulcers.

Two Wistar male rats (weight 150-200q)
After fasting for 4 hours, the alkylated Fc fragment obtained in Reference Example 1 in the amount shown in Table 2 was administered intravenously, and 30 minutes later, phenylbutacin (DWh with 5% gum arabic) was administered at 200 mQ/Kq.
was administered orally. After being left without water for 5 hours, the stomach was removed under ether anesthesia and fixed in formalin. The ulcers occurring in the glandular capsule were determined by the following score, and the summed value was expressed as the 'a ulcer area index.

5core1: Long axis of ulcer 1m 5core2: 1-2m 5core3: 2-3mm score4: 3-4INn score5: 4-5as scorelo: >5am score25: Perforated (al and (bl results are shown in Tables 1 and 2, respectively) 1' shown in 2.

According to Table 1, regarding the effect of alkylated FC fragments on pylorus ligation ulcers, when 10 rlG/Kf was administered 12 times, the ulcer incidence was suppressed by about 74% compared to the control when physiological saline was administered. Furthermore, even with 2 doses of 5#//L7, the dose was lowered to 50%.
% inhibitory activity was observed.

According to Table 2 below, the preventive effect of alkylated Fc fragments on phenylbutacin ulcers is as follows:
m! A significant inhibitory activity of about 73% was observed at the dose of 'j.

Blank space below (2) Pharmacological action alkylated Fcl! The anti-ulcer action mechanism of Li pieces 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 determined by the method of Shay et al. (G
astroenterology 25°906, (1
954)). That is, male Wistar rats (body weight 150 to 200 g) were fasted for 48 hours.
After the pylorus was ligated, the fluid volume, total acidity, and total pepsin activity of the retained gastric fluid for 4 hours were measured. The total acidity is 115ON Na using phenolphthalein as an indicator.
The total pepsin activity was determined by the Anion method using casein as a substrate.
Pharmacol, 13.54. (195B)
). 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 yt/Kg of alkylated Fc1fli fragment was administered to the gastric fluid ostium of the control group,
It was suppressed by about 70% and was further significantly suppressed at 5 rWI/KFI. This tendency was also observed to be similarly suppressed in total acidity and total pepsin activity.

The following margin (I[[) Dosage and administration method Based on the results of the above test, it is preferable to administer the alkylated Fc fragment at 1 to 100 #lff/Aig per day for adults.

This drug can be administered in both injection and oral forms. When used as an injection, it is dissolved in, for example, distilled water for injection before use. The method of administration is intravenous and intramuscular. The drugs used as oral preparations are administered as capsules, tablets, tablets, liposome preparations, or oral liquid preparations. These are manufactured according to methods known to those skilled in the art, such as those described in the Japanese Pharmacopoeia Law.

The agent for treating and preventing gastrointestinal ulcers containing alkylated Fc fragments as a main component of the present invention has extremely low toxicity, and its pharmacological effect is similar to that of a melon, making it extremely effective as a preventive drug for treating ulcers.

Next, reference examples and examples of the present invention are shown. J0 Reference Example 1 [Digestion with plasmin] Sodium azide was added to a 3% solution of 1aG
0 Ing was added and the pH was adjusted to 7.0 Ing using IN NaOH solution.
Adjust to 5. Plasmin is added to a final concentration of 4 cu/i, and digestion is performed at 35° C. for approximately 15 fPf. After treatment 1) Adjust H to 6.5, incubate at 4°C for one period, and remove insoluble matter by centrifugation. Add plasmin digestive fluid (approximately 60i) to 5ephaclex G
-200 column, perform gel a'A treatment,
Undigested globulin (7s) and digestion products (Fab+Fc)
Separate into two parts. This digestion product was then contacted with a column of CM-cellulose (pH 7.0) and the Fab and Fc
Adsorb the fragments.

After washing with the column, O, O + V phosphorus M buffer (pll
Lo) was developed with a solvent containing 0.3M NaCl, and F
Collect ab and Fc fragments separately.

The obtained Fc fragment was dissolved in 0.05 M Tris-HCl buffer (1) H8,2) to a concentration of approximately 2%, and 2-mercaptoethanol was added to a final concentration of 0.75 to 5.25 M. was added to cleave disulfide bonds.

Next, add 015-525M iodoacetic acid to adjust the pH to 80.
After reacting for 1 hour while maintaining the temperature, excess sample was removed using a Sephadex G-25 column. Next, the product was dialyzed against physiological saline, and after sterilization filtration was performed, it was made into a freeze-dried product.

Reference example 2 [Digestion with papain] 2.5% IgG dissolved! (20ml; 0.02M E
Papain 5#+3 was added to DTI-0.05M Phosphorus MM solution (pH 7.5), digested at 37°C for 10-20 minutes, and then cooled with ice water. After cooling, the insoluble matter was centrifuged, and the upper aliquot was fractionated using a G-150 column to obtain a 7s fraction. pH 7.5
-8.0 and treated with dithiothreitol at a final concentration of 0.014M for 2 hours at room temperature, then iodoacetamide was added to a final concentration of 0.2M, and the mixture was reacted for 1 hour under water cooling.

Add this to pH 847) with 0.005M IJ-HCl
+: Cell dialysis was performed, and the resulting crystals were centrifuged. The supernatant was the crude alkylated Fab fragment, and the precipitate fraction was collected as the alkylated Fc fragment.

Example 1 (oral preparation) (1) Alkylated Fc fragment 5.0 mg (2) Fine particles for direct injection No. 209 46.6 mg (Fuji Chemical) (3) iJR cellulose 24.0 ma (4) CM-
Cellulose 4.0 mg (5) Magnesium stearate 0.4 mg This mixed rice was compressed into tablets of 80 mQ each.

Example 2 (intravenous injection) (1) Alkylated Fc fragment 50mQ (2) Butol
Sugar ioomq (3) Physiological saline 10mG After filtering the above mixed solution with a membrane filter, perform sterilization filtration again, dispense the filtrate aseptically into a vial, and fill it with nitrogen gas. It was sealed and used as an intravenous injection.

Example 3 (Capsule) (1) Alkylated Fc fragment 50Q (2) Lactose 935g (3) Magnesium stearate 15g The above components were mixed uniformly, and the mixed powder was placed in two hard gelatin capsules.
00 mg each.

Example 4 (Liposome formulation) Alkylated FC fragments were dissolved in 0.01 M phosphate buffer (1) containing 0.125 M NaCl (87.2) to approx.
Dissolve to a concentration of 0.05%. On the other hand, 0, 5, 10.20% (
Egg yolk phospholipid 10 containing 7-osphatidic acid (W/W)
0m! I was dissolved in 10 ml of chloroborum and a phospholipid film was formed using a rotary evaporator. The above alkylated FC fragment solution 1d was added to this and shaken to form a closed adipose body, and the alkylated FclfiM was incorporated to obtain a liposome preparation.

Patent applicant: Midori Juji Co., Ltd. Representative Patent Attorney Takashi Oyo

Claims (3)

[Claims] (1) A preventive agent for the treatment of gastrointestinal ulcers containing a human 1gG alkylated Fc fragment 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.