JPH0585531B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a gastrointestinal ulcer therapeutic and preventive agent containing an alkylated Fab fragment of human IgG (hereinafter simply referred to as alkylated Fab fragment) as a main component. Until now, the pharmacological effects and medicinal uses of alkylated Fab fragments were unknown. Therefore, it was never used as a medicine. The present inventors have now completed the present invention by discovering that alkylated Fab fragments can serve as a therapeutic and preventive agent for gastrointestinal ulcers. Fab fragments of human IgG have already been reported as constituent fragments of known IgG, for example, as reported by Porter et al. [Biochem.J., 73, 119 (1959)]. This human IgG Fab fragment has a molecular weight of 45,000 and is obtained by digesting human IgG with papain or plasmin.
~50,000 polypeptide chains, and the recovery method was established by Porter et al. The alkylated Fab fragment specified as an active ingredient in the present invention is obtained by cleaving the disulfide bonds of a human IgG Fab fragment and subjecting it to alkylation treatment. A typical recovery method for alkylated Fab fragments according to the present invention is outlined below. 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 Fab fragments are selectively adsorbed, eluted, and recovered. After collecting human IgG Fab fragments, they are treated with a reducing agent to cleave disulfide bonds, and then alkylated to obtain alkylated Fab fragments. As a reducing agent, 2-mercaptoethanol (final concentration 0.75-5.25M), dithiothreitol, or dithioerythritol (final concentration 0.01-0.068M)
etc. are used, and the amount used is a final concentration of 0.01−
This amount is equivalent to 0.068M. Alkylation of Fab fragments is carried out by blocking the SH groups according to conventional methods [Biochemistry, 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 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: -CH ₂
These blocked products such as CONH ₂ can be produced by known methods or similar methods. Next, we will show experiments conducted to confirm the pharmacological actions and effects, clinical trials, acute toxicity tests, dosage, administration method, etc. of the alkylated Fab fragments of the present invention. (1) Pharmacological action and effect The anti-ulcer effect of the alkylated Fab fragment was investigated by causing (a) pyloric ligation ulcer and (b) phenylbutazone ulcer in experimental animals. (a) Shiyai et al.'s method [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.,
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 in diameter)
(above) 5: Perforation The sample 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 Fab fragments obtained in Reference Example 1 in the amounts shown in Table 2 were administered intravenously.
Minutes later, 200 mg/Kg of phenylbutazone (suspended in 5% gum arabic) was administered orally. After being left without food or water for 5 hours, the stomach was removed under ether anesthesia.
Fixed in formalin. Ulcers that occur in the glandular stomach area are
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 Fab fragments on pyloric ligation ulcers is that when 10 mg/Kg was administered twice, the ulcer incidence was approximately 77% compared to the control when saline was administered.
% suppressed. Even at a lower dose of 5 mg/Kg twice, 49% inhibitory activity was observed.

【table】

[Table] According to Table 2, the preventive effect of alkylated Fab fragments on phenylbutazone ulcers is
A significant inhibitory activity of approximately 61% was observed at a dose of 10 mg/Kg of Fab fragment.

[Table] (2) Pharmacological action The anti-ulcer action mechanism of alkylated Fab fragments was investigated. (a) The inhibitory effect of alkylated Fab 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 ligation of 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 (Fab fragment obtained in the 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 the alkylated Fab fragment was administered at 10 mg/Kg, the amount of gastric juice in the control group was suppressed by about 75%, 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 1 to 100 mg/Kg of the alkylated Fab fragment 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 alkylated Fab fragments as a main component of the present invention has extremely low toxicity and exhibits remarkable pharmacological effects, making it extremely effective as a drug 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. Plasmin digested fluid (approximately 60 ml) is injected into a Sephade x G-200 column and subjected to gel filtration to separate undigested globulin (7S) and digested products (Fab+Fc). This digested product was then contacted with a CM-cellulose column (PH7.0) and the Fab and Fc
Adsorb the fragments. After washing the column, 0.01M
Develop with a solvent containing phosphate buffer (PH7.0) and 0.3M NaCl, and collect Fab and Fc fragments separately. The obtained Fab 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 to cleave disulfide bonds. 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 precipitated fraction is an alkylated Fc fragment, and the supernatant is a crude alkylated Fab fragment, and the alkylated Fab is purified by Sephadex G-150 column chromatography, ion exchange chromatography, etc.
Got a piece. Example 1 (Oral preparation) (1) Carbamoylmethylated Fab 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 Fab 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 (Capsule) (1) Carbamoylmethylated Fab 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) Carboxymethylated Fab fragment was dissolved in 0.125M NaCl
Dissolve in 0.01M phosphate buffer (PH7.2) to a concentration of approximately 0.5%. On the other hand, 0, 5, 10, 20% (w/
w) Egg yolk phospholipids containing phosphatidic acid
100 mg of each was dissolved in 10 ml of chloroform, and a phospholipid film was formed using a rotary evaporator. By adding 1 ml of the above alkylated Fab fragment solution and shaking, a closed fat body was formed and the carboxymethylated Fab cross section was incorporated to obtain a liposome preparation.

Claims (1)

[Claims] 1. A gastrointestinal ulcer treatment and prevention agent containing an alkylated Fab 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.