JPS60123428A - Remedy for diarrhea - Google Patents

Abstract

PURPOSE:To provide the titled remedy containing the monoclonal antibody to the diarrhea factor originated from a diarrhea-causing bacteria such as Escherichia coli, and useful as a remedy for diarrhea. CONSTITUTION:The objective remedy contains, as an active component, the monoclonal antibody to the diarrhea factor originated from a diarrhea-causing bacterial strain (especially Escherichia coli). The monoclonal antibody can be produced usually by (1) culturing the diarrhea-causing bacteria separated from the patient of human diarrhea, (2) separating and purifying the cell antigen, or separating and purifying the diarrhea toxin (enterotoxin) secreted from the bacteria, (3) immunizing an animal with the above product as an antigen, (4) carrying out the cell fusion of the lymphocyte of the immunized animal with a myeloma cell (of animal origin) to obtain a hybridoma, and (5) recovering the monoclonal antibody produced by the hybridoma from a culture liquid, etc. The monoclonal antibody is dried and pulverized, and used as a drink by mixing with powdered milk, or is administered orally in the form of enteric preparation for the remedy of diarrhea.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides treatment for diarrhea 11
The present invention relates to a therapeutic agent for diarrhea containing as an active ingredient immunocropurine (monoclonal antibody) which is effective against irradiation, particularly in the form of an 11-stage oral preparation (drinkable preparation and enteric-coated preparation). Even more alarmingly, it is 1' minute compared to all viable bacteria (especially Escherichia coli) that cause diarrhea, and their culture fluids! If purified F.
This is a therapeutic agent for diarrhea, which contains seven IJ-rel antibodies as an active ingredient, prepared by a conventional method using factor 1i as an antigen.

Since the beginning of plasma fractionation, human immunoglobulin, along with albumin, has often been used as an important drug, and today it is indispensable in clinical practice. However, its uses include measles as a measles drug, post-transfusion hepatitis,
It is used for the prevention and treatment of various other infectious diseases, and its administration method is limited to injection.

However, since immunoglobulin derived from human serum or plasma is used as an oral drug,
A manufacturing method for EA/8 preparations has also been established (Japanese Patent Application Laid-open No. 51
-1396 ] No. 77.

It is also known that immunoglobulin given as breast milk in humans and other animals exerts various functions in the gastrointestinal tract of infants who drink it, such as preventing various intestinal infections. Therefore, we focused on this phenomenon, immunized the animal with an antigen corresponding to the antibody we wanted to obtain, collected milk containing a high concentration of the desired immunoglobulin, and made it into powdered milk. A method for producing immunoglobulin for oral administration, which can be given to infants by mixing it with artificial milk after concentrating it, has been invented.
US Pat. No. 3,376,198 (196B).

British Patent No. 8'1 No. 1442283 (+976), Japanese Patent Application Publication No. 51-36326 (197G), U.S. Patent No. 3 +z8
z3o11; (196+)).

Furthermore, animal-derived immunoglobulin administered orally is degraded by the acidity of the stomach and digestive enzymes, and by the time it reaches the intestines, its activity has disappeared.
Up until now, I have been treated without any treatment at stage I-i.
A method for producing a therapeutic agent for intestinal infections using enteric-coated immunoglobulins derived from the milk, serum, or plasma of mammals has also been established (Japanese Unexamined Patent Publication No. 16777-1982).
.

Therefore, Inventor Akiyo et al. decided to use i! Instead of using the conventional technique of immunizing mammals with antigens, we isolated and purified the antigen as a pathogenic factor, and used this to isolate and purify the lymphocytes of the immunized animal and the mini-1-Roma of the animal 11.1. By combining cells with cell fragments, a hybridoma is obtained, which produces a monoclonal antibody against the target antigen.This antibody, or immunoglobulin, is collected and used to treat diseases, which has proven to be extremely effective. In particular, by making such immune globulin into a drinkable preparation or by making it into an enteric-coated preparation that can be administered orally, a much stronger therapeutic effect can be shown compared to conventional immunoglobulin. They discovered this and completed the present invention.

That is, the present invention is produced from a hybridoma obtained by cell fusion of myeloma cells and lymphocytes of an animal immunized with a diarrhea-causing bacterial cell antigen or a diarrheal toxin (enterotoxin) antigen secreted by the bacteria. The present invention relates to a therapeutic agent for diarrhea containing a monoclonal antibody as an active ingredient.

A preferred embodiment of the present invention is a drinking preparation containing an immunoglobulin specific for a diarrheal factor obtained from the culture solution of the monoclonal antibody-producing cultured cells or the ascites of an animal after inoculation of the cultured cells, or the immunoglobulin is prepared in an acidic manner. It is a therapeutic agent for diarrhea characterized by being covered with a film that does not dissolve in bamboo, but dissolves in neutral or alkaline bamboo.

The outline of the manufacturing method of the formulation of the present invention is as follows.

α1-body antigen was produced by culturing Fl/II strain 11ii Mi1^1 (isolated from Hibiscus I, Diarrhea patient-H), and from Il+ bacteria. The amount of secreted F disease toxin (ente+:+l・=l-non) is divided into XI t? Immunization was carried out with +Ml as antigens: 1ilt lymphocytes and minills derived from (animals).
-71111 cells are fused with 1111 I+ (old) to create a hysilitoma. From this hysilitoma, an immunoglobulin (monoclonal antibody) is produced.
Cultivate this antibody? Ii: Collected from fluid or ascites of an animal after hybridoma transplantation. Thus iM
The obtained monoc II-nal lI'L compound is dried and powdered, and this is mixed with, for example, 1151i luk to prepare a drinking preparation. Further, this is formulated by means such as an intestine/H'l preparation.

Immunity-4L1L1. Examples of uj include fluff antigen (CFA/1.CFA/II), which is essential for E. coli colonization and growth in the human intestinal tract. Also used are diarrheal toxins (LT, ST) secreted from the body after colonization.

This fimbrial antigen was discovered by D.G. Evans et al. and is an antigen common to most human-derived diarrheal E. coli [Infecun Yon Ant Inimmunity, 18
．． 330 (1977), and the same magazine, 1st Sat., 63B (1
978)), when diarrheal bacteria enters the intestine orally, it is a colonizing factor that binds to receptors in the intestinal tract, that is, a factor for establishing a foothold for diarrheagenic E. coli, and if this scaffold is not established, diarrheal bacteria will It is believed that the phlegm is released from the body as feces along with the intestinal contents, and diarrhea does not occur.

Similarly, for the treatment of Uno and Pig diarrhea, the pathogenicity in the intestinal tract and the fluff antigen (KO2, K
88), and there is no doubt that fluff is involved as a factor in the onset of diarrhea.

On the other hand, as shown in the test example below, C1" which is a fluff antigen
When monoclonal antibodies against Δ/1 and CF A/II are used as a drinkable preparation or an enteric-coated agent, pharmacologically effective, it appears that these fluff antigen-bearing diarrheal E. coli bacteria attack colonize and proliferate in the intestinal tract. It has been shown that these preparations are highly effective in inhibiting
It suggests sex.

On the other hand, in human-derived diarrheal E. coli, there are two types of enteroxins that make heat resistance W.
, Ii (L i', ST), and it is known that there are strains that produce both, and strains that produce each individually [Journal of
Medical Cyclobiology, 3,387 (19
70)). The mechanism of action of these toxins is similar to that of cholera toxin; when the toxin binds to the receptors of small intestinal atrophic N-J bidermal cells, it activates azonyl cyclase in the cell membrane, resulting in cyclic AMPfi in the cells. The degree will increase. As a result, increased secretion of chloride ions and inhibition of absorption of 1-lium ions occur in mucosal epithelial cells, leading to excessive secretion of water, resulting in what is called a state of water retention in the intestinal tract, i.e. diarrhea symptoms. will be presented.

Therefore, as shown in the test examples below, when we created monoclonal antibodies against these diarrheal toxins and examined their pharmacological effects in drinking and enteric-coated formulations, we found that High effectiveness in preventing water retention was demonstrated. In other words, it was inferred that these preparations neutralized diarrheal toxins secreted by E. coli.

On the other hand, immunoglobulins (monoclonal antibodies) can be obtained from hybridoma culture fluid in vitro or from animal ascites after hybridoma transplantation in vivo.
Purification methods from serum or plasma can be used to obtain [transfusion, 6
．． 146 (1966), Journal of Biological Chemist Riichi, 234.2645 (1959
), Biochemistry Eto Biophysics Acta, LL!
L., □, 107 (1970)). Furthermore, as an industrially suitable method, purified immunoglobulin can be separated from 1A by stepwise ammonium sulfate fractionation, which can be further separated by ion exchange chromatography, gel filtration θ2, etc.

It is preferable that the immunoglobulin thus obtained is undized by a method such as freeze-drying. This is then mixed with an excipient such as powdered milk to form a drinking preparation. Furthermore, by covering this powdered immunoglobulin against each antigen with an enteric coating, it is stabilized, passes through the stomach, reaches the intestines, and the coating decomposes, allowing the contents to be stabilized. It can be a formulation in which the substance is released.

This method of intestine/81P treatment can be applied to any conventionally known method by selecting the conditions.
C-kill. The conditions include heat treatment, use of organic solvents, and other conditions that do not cause protein denaturation.
) Shitea? J.

4 for this invention? Enteric film-forming materials that can be used (for example, methyl methacrylate 1, methyl methacrylate I'l'J combination, polyvinyl acetate phthalate, cellulose acetate succinate 1-, Examples include hydroxypropyl methylcellulose phthalate, cellulose acetate phthalate, cellulose propionate phthalate, and methylseno1) 1-cose phthalate. These substances are all organic solvent-soluble/dissolvable film coating agents, and can be dissolved in appropriate amounts in alcohol, acetone, etc., and sprinkled onto solidified immunoglobulin.

The enteric coated immunoglobulin preparation can be prepared by filling purified dry powdered immunoglobulin into commercially available capsules made of gelatin matrix, and coating the capsules with the enteric coat-forming substance described above; Preferred forms include enteric-coated small particles such as microcub nano-mini pellets.

When made into microcapsules, they can be mixed with powdered milk, for example. In this case, care must be taken to control the temperature when adding hot water. To control the temperature more easily, add hot water to the mill, then add microcapsules to the 0, 1+, and 2 wafers whose temperature is below a constant temperature (for example, 40°C) to maintain a uniform temperature. ! ! For example, infants can drink it from a feeding bottle or the like.

The manufacturing method for Minibeleno I is as follows: It is preferable to prepare an intestinal lhing preparation. As, human xozo 1.1 bicellulose, hIII xyzo I nopyl methyl cellulose, polyheal pi 1! lydone, vinyl vinyl 1:I liton-hinyl acetate--1-conjugate, methylcellulose, pyro 1 kinnie f-le 1! 11-su, carpoxomethylcell li+-
Suritoru J1, Polyhinyl Alnylene 1-L, Arahia 1
), nato-(,9) After coating, the above-mentioned organic solvent-soluble enteric film 21-taining agent may be coated on the organic film I, and the like.

This coating method is as follows:
Intestines/94! For l:::I-ting, 50℃I
J, below, preferably at a temperature below 30°C, and coated with 18 parts of the coating agent in an organic solvent.

Similarly, as step 11 of the sub-coating method, 5
For example, coating with a solution of a coating agent dissolved in water at a temperature of 0° C. or below, preferably 3 ()° C. or below. In addition, a solution in which a coating agent is dissolved in an aqueous solution containing a small amount of an organic fH medium also produces cj+ with iiJ ability.

The enteric-coated immunoglobulin preparation thus obtained is administered as a capsule, mini-bereyl, etc. When this drug is administered intravenously, the capsule is tightened in the intestine and a specific immunoglobulin is released, and the pharmacology of this drug is It exerts its effect on the right side in the intestinal tract.

The dosage of the monoclonal antibody varies depending on body weight, age, severity of diarrhea, dosage form, etc., but for example, for diarrhea in infants,
The dosage may be slightly different.

In addition, this monoclonal antibody can be used in Example 2 and a
＋(It is clear from Experimental Example 2) that it is extremely safe.
.

The treatment agent for dysentery J+i of the present invention is much more effective than those derived from milk, serum, or plasma, which are used for conventional mammalian immunity, and Diarrhea I
In a trial different from the treatment method using therapeutic agents, etc., which inhibits E. coli, the ``4! It is one thing to demonstrate one's medicinal skills, and it is extremely
It is said to be highly useful.

Example I Utsuk1. ! -Creating null antibodies-'
((11) CFΔ/I pili ia, Escherichia coli (11-, ,
-1,04Fl 7 stocks), ■, Su, Mata C FΔ/11
Fimbriae are derived from Escherichia coli (53402, △-1 strain) and are grown on CF∆ agar medium.
After checking the filtration with 4.1 (anti-K), the cells were cultured in large quantities, and the resulting bacterial cells were isolated and purified by the method of Evans et al. j-9, 727 (1978
), and the same magazine, j,-i,,,525 (198
0) ].

In addition, LT, a diarrheal toxin, is derived from Escherichia coli (Ii-1 (14
07 strain), S i'' was isolated and purified from (53402, △ 1 strain) by the method of Kohans et al. using each of these J & nutrient solutions as starting materials [Infekunyong An1 Inmunirai, , -j-0-, +0
10 (1974), and the same a: U, -2-'q, .
978 (1979)).

After immunizing 13alb/c mice with each of these four antigens, 111 cells were isolated in the lungs, and according to the method of Köhler et al.
Gore and NSI/5l) 2 mini 1 coma cells and polyconytylene glycol are used for cell fusion.
From the experiment of li:11, 174 Nori I corns were grown in Hutt's medium or I corn (when CFA/I was used as an antigen). The activity was checked by enzyme antibody method using alkaline fumes facuse as a target.

Among the hybridomas that produced antibodies against each of the +J'11 antigens of C5, the clones that produced the most W were blistane (treated 13a11+/c, transferred to the peritoneal cavity of mice, 11G, 1()~ Table 1 shows the results of measuring the antibody titer in mouse ascites after 14 days using the f+'l antibody method.

In addition, Y-111111iq and a
Test method using mouse mouse 7! (The results are shown in Table 2.
It is.

(Table 1) *Antibody titer was determined by adding serially diluted ascites to a plate coated with 5 μg of each purified antigen, and then adding anti-mouse Ig to a plate coated with 5 μg of each purified antigen.
The enzyme antibody method was used to measure G-Ali's strength using Fumeshupkuse reagent. The antibody titer is indicated as the maximum Ir (+ dilution rate) of mouse ascites, which exhibits the same regurgitant intensity (absorbance) as control 711.

(Table 2) * Shows the liquid crystal dilution rate of each mouse ascites that neutralizes and inhibits 1 unit of toxin. That is, for L'I', 50%♀1 of mouse adrenal gland cells (Y-j) [1 cell degeneration is the target, S'
F is the intestine weight/Iki weight after administering a mixture of ST and ascites to suckling mice intravenously for 1 hour.
It became a mark.

Furthermore, the activities of these four monoclonal antibodies are
To confirm that it is antigen-specific, the test was performed by changing the combination of antigens), anti-CF△
Ascites containing /1 antibody is a fluff antigen C F△/11
, K2S (1 quintillion anti-pig-derived E. coli strains) +<!
] 9 (fluff antigen of E. coli derived from bullfinch) did not react with ascites diluted 10 times. In addition, anti-CF A/
Ascites containing 11 antibodies also c +r△/1, K 88, K
In the ascitic fluid diluted with IF 1-fold, there was vomiting with the 94+ line antigen. Additionally, anti-1-T, fI
c S゛1゛1゛゛L-containing ascites of each mouse was also immunological;
There was no vomiting or vomiting at all when diluted 10 times. That is, it can be said that each monoclonal monoclonal l'Ll of 4 insect hair f1, i< is Lb with 4Nv-like immunity to the immunogen. In addition, 11f monoclonal 1
] L body, and (,1?8/1, CF8 to II fimbrial antigen 1, 3-1; l: l I4 G 2 to class S-→ 4) Su, 1. Diarrhea 1! l
';! j Cumulative L T, ST 1-9 is 11
! G l (1) 41 It belonged to Zokunos.

Example 2 Mouse ascites after transferring Nori 1-/
and high-solid 1-ma were treated with liN using Solasni 1], using this 1'&l-yen as starting material - (,5()% ammonium sulfate precipitate, D
I! A purified immunoglobulin fraction was obtained by cellulose gel filtration and Sephadex G-200 gel filtration, and freeze-dried to obtain an immunoglobulin powder. The recovery rate of immunoglobulin in this purification process is 5.
It was 5-65%. After one week of oral administration of 40m8 of this product to 20±2g mice, no abnormalities were observed. Then, 14.5 g of commercially available powdered milk was added to 0.5 g of immunoglobulin powder and mixed uniformly to prepare a drinking preparation.

On the other hand, to 0.5 g of immunoglobulin powder was added 1.4.5' ++ base, which was a mixture of 55% Niscudi, 43.8% Ahyal, and 1.2% magnesium stearate, and added it to 0.5 g of immunoglobulin powder. I put things in No. 3 capsule I 20 :j
: Fill 10 mg each, 1 course of 1 to 8% acetate phthalate, 5 methyl ethyl getone [)]
%, methylene chloride 35%, and triacetin 1% to make it enteric coated): 1-
Enteric-coated turnip of each immunoglobulin (seven clonal antibodies) was applied. I made a ru.

The recovery rate of immunoglobulin in the product is 90 to 95% even in the case of -1, when the filling amount is 1 Ofl.

Next, for six separate trials, each immune patient who was HHI
: 1 zoline) book (1.5g 'J' each with 70% corn starch, °) 'hycel 20%, carpoxomethyl l! /l/ l:l -ska/l/ '7um5
05Q) 5%, Hill: I Kinobu II Hill I? Base (III) C) 5% of base 2I! lJ
After adding water and kneading, it becomes spherical! Please granulate fI grain 6 and dry it in the air! l\i line 0.5mm (7)'
Approximately 2.5g of pfj grains were weighed 1!4 times. This includes polyvinyl vinyl 1
” Ridon (PVP) +1 +1%, I+ 11 C2
Dissolve the powder in water (mixed at a ratio of 1%). After applying ``Tabni'' coating, a film consisting of 1.10% Cell II SpII bionate phthalate, 49% acetone, 40% isopropanol, and 2% triacetin was removed and each enteric-coated mini-vellet ``ζ'' was applied. f'lta.

Phil J.: The increase in total weight after I-ting is about 40
J) in %.

In Test Example 1, an animal test was conducted to confirm the pharmacological benefits of a drinkable immunoglobulin preparation and an enteric-coated immunoglobulin preparation containing a monoclonal antibody against F-disintegrating factor derived from intestinal obstruction. Example 2
enteric anti-CF A/1 immunoglobulin mini-pellet obtained by the method [a], enteric coated 1-1 anti-CF A/11 immunoglobulin mini-pellet 1 (b), normal mouse blood lP1 immunoglobulin mini-pellet, 1.[C] are each 3 (l m
, g (4.2 mg as immunoglobulin amount), and enteric-coated firn, and mini pellets without drug (a', b', c':l (4.2 mg as immunoglobulin), respectively) were used. . Furthermore, in drinking formulations, anti-C
Powdered milk containing FA/I immunoglobulin [d], Powdered milk containing anti-CF A/II immunoglobulin [e]
], Serum Immunization of Serum Mouse 1: Powdered milk (f) containing I purine for 12911/! (each immunoglobulin amount was 4.2 mg).

Three 3-day-old rabbits were fed -8Y, and the respective feeds were orally administered to each group 6 hours or 1 hour before administration of the challenge bacteria. (6 hours ago: a, b, c, a', b',
c', 1 hour ago: d,,e,,r).

After that, one i 0 + of diarrheal E. coli was orally administered in an alkaline buffer, and after 24 hours, the L, 1 and middle portions of the small intestine were removed and the E. coli age was measured using a selective medium. Got the results. In addition, 61 of the inoculated bacteria
Inoculate 1 seed grown on the medium.
Using an agglutination reaction using anti-+m serum obtained from i'e immunization, we used CI2'j as a sample.
It has a strong infection prevention effect against diarrheal human intestines that corresponds to the specificity of IJ purine! 1!9))) (qualitative inhibition of the intestinal tract) was observed. In addition, enteric-coated filtration and anti-CF immunoglobulins are also effective.
It was tolerated, but it was weaker than the sample that had been cowed. On the other hand, in the case of a drinkable preparation made by mixing immunoglobulin with milk, the 'A' fruit was smaller than that of the enteric coated product, but the 'A' fruit was found to suppress the growth of the inoculated bacteria. In addition, in all formulations of 1ζ, immunoglycium II purine derived from normal animals not subjected to immunotherapy 1'1 did not show any effect.

(Margins below) (Table 3) *After inoculation of challenge bacteria to animals without sample administration (oral, 1+16
) shows the number of inoculated bacteria in the small intestine after 2 hours.

Next, in order to investigate the pharmacological effects of the water retention rate in the intestines caused by enteric toxins, enteric-coated antimicrobials were used as before.
'l'Immun13J globulin minibellet (a), anti-ST
Immunogen 1゛Jzolin Minipereso 1 [b], normal mouse serum immune clobulin Minipereso 1 (C) were added at 3 (ln
u+ (immunoglobulin amount: 4.2 mg), and 22 mg of mini pellets (a', b', c', respectively) without enteric film agent (a', b', c', respectively)
4.2 mg) was used as the amount of purine. Furthermore, the drinking formulation contains anti-CF A/I immunoglobulin15)
Milk [d], containing anti-CF/U immunoglobulin 45
) milk [e], normal mouse serum immunoglobulin containing 1-1 purine J'51 milk (f), respectively 129IIIF
X (4.2 mg as Immune II Purine M) was used.

Immediately after challenge, each sample was orally injected into each group of three 1J-gi mice aged 311 [Io]. Bacterial attack: [106 pieces of ζ-disintegrating Escherichia coli were injected into the mouth,
Sushi, remove the intestine after 24 hours (1), then remove the intestine contents and measure the weight, then measure the weight of the remaining intestine after removing the contents, calculate the ratio, and calculate the water retention rate. The results shown in Table 4 were changed.In other words, the immunoglobulin preparation, which responds to the toxin produced and secreted by the inoculated bacteria, suppressed the accumulation of water in the intestine.The drinkable preparation was enteric-coated. The effect was slightly smaller than that of .

(The following is a blank space) (Table 4) Table 4 shows the ratio of monthly cargo weight 81 (g)/lifted weight i1: (g) of animals 24 hours after inoculation with the challenge bacteria.

Test Example 2 Escherichia coli-derived diarrhea obtained in Example 2 4! Immunoglycobulin against ll factor (4 types of drinking preparations each containing monochrome/14 几(Thursday), Sokoma, Rao) 2φ
Mixed dosage formulations, and 4 types of enteric coated minivere each.

l・, the safety test of their equal amounts mixed preparation was carried out on body weight 20±
A mouse weighing 2g was continuously thrown for 31 minutes and observed for one week, but no damage was observed.

Patent Applicant Mi]-rijuji HJ-"-Ito 15 Ne City -1-E 'n, 2F (
Spontaneous) ■ The person behind the incident, Showa 53) 4'! j i'l request No. 229485υ2,
Title of the invention 1. Diarrhea 11. Therapeutic Agent 3, Sleeve II': '? Relationship with c l'rugo'old'1 Patent applicant's name (name) Mi1-rijuji4 Co., Ltd., agent ■
541 Address: 1, 4-chome, Hirano-cho, Higashi-ku, Osaka [53-3].

-,1--Life flat! r-cho 40 fi-■Telephone (06
) 2271156 6, Detailed description of the invention-1 7.7 City 11
- Contents (11 Akira ■ 1 ¥ 11 page 15, correct 1-alkari in the last line to 1 alkali-1. -1

Claims (3)

[Claims] (1) A therapeutic agent for diarrhea whose active ingredient is a monoclonal antibody that causes diarrhea. (2) Claim No. (2) in the form of an oral Jj preparation
The agent for treating diarrhea described in section 1). (3) The therapeutic agent for diarrheal disease according to claim (1) or (2), wherein the first bacterium is Escherichia coli and the diarrheal disease is human diarrheal disease.