JPS6013716A - 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, and improved medicinal effect such as inhibitory activity against secretion of acid in the stomach, inhibitory activity against total pepsins, etc., containing an alkylated Fab fragment of human IgG as an active ingredient. CONSTITUTION:An alkylated Fab fragment of human IgG is as an active ingredient of a remedy and preventive for ulcer of digestive organs. The alkylated Fab fragment is obtained by scissoring disulfide bond of Fab fragment of human IgG, alkylating it. It is found that the alkylated Fag fragment has low toxicity, improved antiulcer action, and 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 treatment and prevention agent for gastrointestinal ulcers, which contains an alkylated Fab fragment of human TgG (hereinafter simply referred to as alkylated Fab fragment) as a main component.

Hitherto, the pharmacological effects and medicinal uses of alkylated Fab fragments were unknown. Therefore, it was never used as a medicine. This time, the present inventors have discovered that alkylated Fa
The present invention was completed by discovering that fragment b can be used as a therapeutic and preventive agent for gastrointestinal ulcers.

The Fab fragment of human 1aG has already been reported as a constituent fragment of known IgG, for example, as reported by Porter et al.
iochem, J., 73.119 (1959)
). The Fab fragment of this person [oG is human-derived I (
It is a polypeptide chain of 145,000 to 50,000 molecules obtained by decomposing IG with papain or plasmin, and its recovery method was established by Porter et al.

Alkylated F specified as an active ingredient in the present invention
The ab fragment is obtained by cleaving the disulfide bonds of the Fab fragment of human IaG and subjecting it to alkylation treatment.

A typical method for recovering alkylated 1''ab fragments according to Honmaru Akira is outlined below.

A solution containing IQG (protein't 1 degree 2-10%) was added to p
Adjust to H6-9, 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 IFG and digestion products are separated by gel filtration treatment. The products of digestion are ion exchangers (CM-cellulose and DE
Chromatography is performed using AE-cellulose) to selectively adsorb, elute, and collect Fab fragments of human IQG.

After collecting the Fal+ fragment of Person 1 (IG), it is treated with a reducing agent to cleave disulfide bonds, and then alkylated to obtain an alkylated Fab fragment.

As the 17 base agent, 2-mercaptoethanol, DidiA
Threitol, dithioerythritol, etc. are used in an amount corresponding to a final concentration of 0.01-0.068M.

Alkylated Fab fragments are achieved by blocking the SH groups according to conventional methods (Bto-chem
istry, 7 (5), 1950-1958 (
1968)). Examples of such groups include the following.

■ Lower alkyl group: methyl, ethyl, n-propyl, etc. ■ N,N-di-lower alkylcarbamide-lower alkyl I: N, N-diethyl-carbamidomethyl ■ Lower alkoxycarbonyl group: engineered toxycarbonylmethyl, ethoxycarbonylethyl etc. ■ Carboxy-lower alkyl group: carboxymethyl, carboxyethyl, etc. ■ Cyano-lower alkyl group, dianmethyl, etc. ■ β-amino-lower alkyl group, CH2CJNH2
etc.■ Benzoyl-lower alkyl CHc006H5
These blocked products can be manufactured by means known per se or by means equivalent thereto.

Next, the pharmacological action and effect of the alkylated Fab fragment of the present invention,
The following describes clinical trials, acute toxicity tests, and experiments conducted to confirm the administration and method of administration.

(1) Pharmacological action and effect In experimental animals, (a) pylorus ligation ulcer and (b) phenylbutacin ulcer were induced, and alkylated Fabll
The anti-ulcer effect of the i-piece was investigated.

(a> Shai et al.'s method (aastroentero+
ogy.

Medium, 43. (1945)), a pylorus ligation ulcer was created. That is, male Wistar rats (body weight 200
After eating ~250g for 24 hours, the stomach was removed under ether anesthesia, and the ulcer that had developed in the forestomach was treated with Marumi.
) et al. (J, Takeda Res.

Lab,, 15.85. (1970)), the following ulcer index was used for evaluation.

〇2 Normal 1: Erodion or bleeding spots 2: 10 or less small ulcers (1 m or less in diameter) 3: 10 or more small ulcers or 10 or less medium ulcers (2.4 mounds in diameter) 4: 10 or more Medium or large ulcer (4 shoes or more in diameter) 5: Use the specimen obtained in Reference Example 1 as the perforated specimen, dissolve it in sterile physiological saline, and perform the following procedures in Table 1 immediately after ligation and after 8 hours.
The legs shown in Figure 2 were administered intravenously twice.

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

26.471 (1976)) to create phenylbutacin ulcers.

2 male Wistar rats (weight 150-200J)
After fasting for 4 hours, the alkylated Fab fragment obtained in Reference Example 1 shown in Table 2 was administered intravenously, and 30 minutes later, phenylbutazone (suspended in 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. For ulcers occurring in the glandular stomach region, the following scores were determined and the summed value was expressed as an ulcer index.

5core: Ulcer long axis 1 body 5Core2: 1~2I+111 score3: 2~3g 5core4: 3~4m 5Core5: 4~5Mn 5core10:>5# 5core25: Perforated (al and (11) results are shown respectively) 1 and Table 2.

According to Table 1, alkylated Fab[! Regarding the effect of Li pieces on pyloric ligation ulcers, when 10η/Ky was administered twice, the ulcer incidence was suppressed by about 77% compared to the control when physiological saline was administered. Further lowered dose 5 11I/892
Even after multiple administrations, 49% inhibitory activity was observed.

According to Table 2 in the margin below, the preventive effect of alkylated FabljIi pieces against phenylbutazone ulcers is greater than that of alkylated Fab
Fragment 10@/Kf! Approximately 61% of the inhibitory activity was observed at a dose of .

Margin below (2) Pharmacological effects We investigated the anti-ulcer action mechanism of alkylated Fab fragments.

(a) The inhibitory effect of alkylated Fab fragments on gastric 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
astroenteroloqV 26°906, (1
954)). That is, male Wistar rats (body weight 150-200 g) were exposed to sound for 48 hours.
Ligation of the pylorus @ 4 hours of retained gastric fluid, the fluid!
, total acidity, and total pepsin activity were measured. The total acidity is +15ON Na using phenolphthalein as an indicator.
The total pepsin activity was determined by the Anson method using casein as a substrate.
Pl+armaco1. , 13.54. <195
8>). The specimen (Fab 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 Fab fragment was administered, the amount of gastric fluid in the control group was approximately 7
It was suppressed by 5% and was also significantly suppressed at 5tyt/Kg. This tendency was also observed to be similarly suppressed in total acidity and total pepsin activity.

In the margin below (I

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. When used as an oral preparation, it is administered as a capsule, tablet, powder, liposome preparation, or oral liquid preparation. these are,
For example, it is manufactured according to a method known to those skilled in the art described in the Japanese Pharmacopoeia Law.

The agent for treating and preventing gastrointestinal ulcers containing an alkylated Fab fragment as a main component of the present invention has extremely low toxicity and does not exhibit significant pharmacological effects, making it extremely effective as a drug for treating and preventing ulcers.

Next, we will discuss reference examples and examples of the present invention.

Reference Example 1 [Digestion with plasmin] 1 (60 rRg of sodium azide was added to a 3% solution of IG (60 aff), and the mixture was digested using IN NaOH solution) H
Adjust to 1.5. Plasmin at a final concentration of 4 cu/-
Digestion treatment was performed at 35°C for about 15 hours. After treatment, the DH was corrected to 6.5 and the temperature was increased to 1 at 4°C.
After incubation for an hour, remove insoluble matter by centrifugation. Add plasmin digestive fluid (approximately 60m) to 5ephadex G-2
00(7) Column 2ff is entered, gel filtration is performed to separate undigested globulin (7s) and digestion products (Fab and Fc).
) and separate. This digested product was then contacted with a column of CM-cellulose (pH 7,0) and the Fab and F
Adsorb the c fragment.

After washing the column, add o, o+M phosphate buffer (pl
llo) was developed with a solvent containing 0.3M NaCl,
Collect Fab and Fc fragments separately.

The resulting Fab fragments were dissolved in 0.05 M Tris-HCIM.
It was dissolved in a buffer solution (pH 8.2) to a degree of f of about 2%, and 2-mercaptoethanol was added to a final concentration of 0.75 to 525M to cleave disulfide bonds.

Light T O, 75-5.25 MP H5” FM add;
A, l) After 111ffi1 reaction was carried out while keeping H 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 was performed, it was made into a freeze-dried product.

Reference Example 2 [Digestion with papain] 2.5% solution of IqG (20ml: 0.02M E
Papain and 5IRy were added to DTA-0.05M phosphate buffer (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 Sephadex G-450
It was fractionated using a column to obtain a 7s fraction. This pH7,
Dithiothrei 1 to 5-8,0 with a final concentration of 0.014M
The mixture was treated at room temperature for 2 hours, and then iodoacetamide was added to a final concentration of 0.2M, and the mixture was reacted for 1 hour under water cooling.

This was dialyzed against 0.005 M l-Lis-HCl at pH 8, and the resulting crystals were centrifuged. The precipitate fraction was an alkylated FC fragment, and the supernatant was a crude alkylated Fab fragment. Purified alkylated Fab fragments were obtained by Cef1dex G-150 column chromatography, ion exchange chromatography, etc.

Example 1 (oral preparation) (1) Alkylated Fal+ fragment 5.0m (]<2)
Fine particles for direct hitting No. 209 46.6m.

(Fuji Chemical) (3) Crystalline cellulose 24.0mg (4) CM
- Cellulose 4.0 mg (5) Magnesium stearate 0.4 mg This mixed powder is compressed into tablets, each tablet has an 80
It was made into a tablet of m.a.

Example 2 (intravenous injection) (1) Alkylated Fab fragment 50mG (2) Glucose ioomq (3) Physiological saline 10mQ After filtering the above mixed solution with a membrane filter, sterilization filtration was performed again. The filtrate was aseptically dispensed into vials, filled with nitrogen gas, and sealed to give an intravenous injection.

Example 3 (Caprel agent) (1) Alkylated Fab fragment 50C1 (2) Lactose 935Q (3) Magnesium stearate 15Q The above components were mixed uniformly, and the mixed powder was placed in hard gelatin capsules.
00 mG each.

Example 4 (Liposomal formulation) Alkylated Fab fragments were prepared in 0.125M NaCl.
001M phosphoric acid containing! About 0 to l buffer solution (DH7,2)
Dissolve to a concentration of 5%. On the other hand, 0, 5, 10.20% (W
/W) Egg yolk phospholipid containing phosphatidic acid 100
sy was dissolved in 10-chloroform, and a phospholipid film was formed using a rotary evaporator. Add 1 part of the above alkylated Fab fragment solution to this,
By shaking, closed fat bodies were formed and alkylated Fab fragments were incorporated to obtain liposome formulations.

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

Claims (3)

[Claims] (1) A gastrointestinal ulcer therapeutic and preventive agent containing an alkylated Fab fragment of human IQG 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 digestive organ i1 according to claim (1), which is in a liquid form for intravenous, intramuscular or oral administration! Il
! Treatment preventive agent.