JPH01268645A - Agent for suppressing schwartzman reaction - Google Patents

Abstract

PURPOSE:To provide the subject suppressing agent containing an active component comprising a component effective in inactivating tumor necrosis factor (TNF) or removing the factor from the circulatory system. CONSTITUTION:The objective agent contains, as an active component, a sub stance effective in inactivating TNF or removing the factor from the circulatory system. The substance is preferably an anti-TNF antibody, especially an anti- TNF neutralizing antibody. The administration of said agent suppresses or mitigates the systemic shock or local organopathy and dermatopathy caused by Schwartzman reaction. It is administered at a rate of <=20mg/kg, preferably 0.1-15mg/kg per day. The above antibody can be produced in an animal body immunized with TNF or produced by a hybridoma cell.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial Application Field The present invention relates to an inhibitor of the Schwartzmann reaction induced by endotoxin and the like.

(b) Background of the invention The Schwartzmann reaction is caused by endotoxin, etc.! e
It is the name of a reaction accompanied by tissue necrosis, bleeding, etc. induced by substances [Sadaaki Iwanaga et al., eds., [Endotoxin], Ishiyaku Publishing Co., Ltd.
1983. 245-258] 1 The local cutaneous Schwartzman reaction was first reported by G. Shwartzman [G. Shwartzian, Pr.
oc, Soc, Exl], Biol, Mecl, 255
607561. (19281J.

The Schwartzmann reaction is generally performed by bacteria, etc.
It can occur in solids due to dead bacteria, bacterial arm fragments, etc. Endotoxemia may cause endotoxin shock and result in death, but in this case, systemic Schwartzman reaction and multiple organ failure due to Schwartzman reaction play an important role, and Schwartzman reaction is In addition to the systemic shock caused by endotoxemia, it is also a disease in which organ function is lost due to symptoms such as hemorrhagic necrosis in various organs within the body, such as the liver, pancreas, and adrenal glands. It is assumed that the Schwartzmann reaction is involved in severe hepatitis, acute pancreatic necrotizing nephritis, etc. [Advances in Endotoxin Clinical Research, edited by Toshitsugu Oda and Yuo Yamamoto, Yokosha, 1985].

Although much research has been carried out on the mechanism of the Schwaramann reaction, the identity of the mediator and the reaction mechanism have not yet been clarified.

(C) Purpose of the Invention The present inventors have conducted intensive research with the aim of suppressing the Schwartzmann reaction that occurs in a wide variety of diseases. It is a derivative of suppressing the Schwartzmann reaction.

(d) Summary of the Invention Specifically, the present invention is a Schwartzmann reaction inhibitor containing as an active ingredient an agent having the activity of nullifying or removing TNF from the circulatory system.

The present invention will be explained in detail below.

The substance having the activity of nullifying TNF or removing it from the circulatory system used in the present invention is not particularly limited as long as it has that effect, but examples include rats,
Mouse, rabbit, goat, goat, sheep.

Polyclonal or monoclonal antibodies produced by various animals or animal cells, such as horses, can be utilized. These can be immunized with TNF by methods well known to those skilled in the art, and antibodies produced in the animal body can be obtained by conventional methods, or can also be produced by hybridoma cells. Jr. mela antibody may be used.

Anti-'1' N F as mentioned above is one that has the activity of nullifying 'l' N F or removing it from the circulatory system.
I? It is preferably an antibody, and more preferably an anti-'T' NF neutralizing antibody.

The Schwartzmann reaction inhibitor used in the present invention, which has an active ingredient that has the activity of nullifying 'I' N F or removing it from the circulatory system, is administered systemically before or after the Schwartzmann reaction occurs. Accordingly, it can be used as a prophylactic or therapeutic agent. In this case, there are no particular limitations as long as the drug is administered systemically, for example, intra-YprR administration! lJ intravenous administration,
Examples include intraperitoneal administration and intramuscular administration. In addition, it is thought that one of the mechanisms of action of the drug is that it is transported to each local tissue after systemic administration and exerts its effectiveness, so local administration at the site of the Schwartzmann reaction can also achieve the desired purpose to some extent. It seems that we can reach it. Furthermore, in order for the drug to function effectively, an appropriate amount of the active ingredient of the drug must be present in the blood and supplied to each tissue during the progress of the Schwartzmann reaction, or an appropriate amount must be present in the local tissue that causes the Schwartzmann reaction. It is necessary that an amount of the active ingredient of the drug be present. In other words, as long as these conditions are met, the divine method of administration is possible. Systemic intravenous administration of the drug is preferred.

The dosage of this drug is 20 μ/'k [r/day or less, preferably 0.1 to 15 μ/k+r/day].

The drug of the present invention can be applied in the form of a composition mixed with other ingredients as long as it maintains the activity of neutralizing or removing 'I' N F from the circulation.

When this drug is used as an injectable composition, excipients include amino acids, saccharides, cellulose derivatives, polyvinyl and lolidones, 5 organic acids, and mW metal compounds.

Examples of amino acids include glycine, arginine, alanine, and pharmaceutically acceptable salts thereof. Sugars include mannitol, inositol, xylitol, and lactose.

Glue: Examples include l-su. As a cellulose derivative, carboxymethyl cell 1 course sodium.

Examples include methylcellulose. Min 'j' for polyvinylpyrrolidones! L10.000~1,000
,000 polyvinylpyrrolidone.

Examples of organic acids include ascorbic acid, citric acids, and salts thereof. @ Machine combination! Examples of [r() include sodium hydrogen phosphate, sodium hydrogen carbonate, and sodium acetate.

Generally, one type is selected from these compounds, or two or more types may be mixed.

These excipients also include glycine, arginine.

7'''ranine, pharmaceutically acceptable salts thereof, mannitol, inositol, xylitol, and the like are preferred.

Preferred solutions for dissolving excipients include distilled water for injection, physiological saline for injection, and Ringer's solution for injection.

The injection form is preferably 0.2 parts by weight of the drug.
~200 parts by weight are used.

Sodium pyrosulfite is used as a stabilizer! -Antioxidants such as ascorbic acid; Antioxidants such as EI) TA, 100-glycol agents, and the like. Examples of the surfactant include nonionic surfactants such as polysorbate and polyoxyethylene derivatives. 6 Examples of the tonicity agent include sodium chloride.

As analgesics, benzyl alcohol, xylocaine,
Examples include professional skills.

Parabens and chlorobutanol are preservatives.

Benzalkonium chloride, Thimerosal (Thimcro
sat), etc.

Examples of the emulsifier include sodium salts of citric acid, acetic acid, phosphoric acid, and the like.

Examples of specific compositions of the formulations applied in the present invention are as follows.

Composition Example 1 (in 10 ml) Composition Example 2 (in 10 ml) Composition Example 3 (in 10 ml) (e) Examples The present invention will be explained in more detail with reference to Examples below. It is not limited.

Example 1 (Preparation of anti-'I' NF jic antibody) The anti-'f' NF antibody used in the present invention is based on the previously filed Japanese Patent Application No. 162233-1982 (filed July 111, 1988). Monoclonal antibodies and hybridoma cells were produced by the method described in 1, that is, they were produced as follows.

1 ward, 2 violet 1 E. coli into which the human '[' NF gene expression vector had been introduced was cultured to promote the production of the HiI-T NF protein. After collection, E. coli was disrupted using ultrasound, and the resulting suspension was used by 5hirai et al. [T, 5hirai at
at, Nature, 313, 830 (1985)]
Purified by DEA and EScpharosa chromatography according to the method of 0! The 1NF content in the fil purified product was about 30%. Human T'NF
by mouse.

The 'I' NF fraction (50 ~
100 μf) subcutaneously at 2 week intervals.

Four days after the final immunization, the lungs were removed and used for cell fusion.

State JJL gold cell fusion was performed according to standard methods. That is, a cell suspension was prepared from a lung aseptically removed through a mesh, washed three times with RPM11640 medium, and then P3-X63-Ag8-Ul cells (P3
(sometimes abbreviated as R1) [D, E, Ye I
Current Topics in lli
crobiologyand IIDlunology
81, 1 (1978)] and approximately 1 = 1 to 5:
Mix at a ratio of 1:1 and 50% polyethylene glycol into the pellet after centrifugation. rt PMI-164
1 ml of Offi solution was gradually added and the centrifuge tube was slowly stirred for 1 minute to perform cell fusion. Furthermore, RPMI-1
640 medium was gradually added over time until finally 10
It was set as m1. After centrifugation, the pellet was placed in RPM11640 medium containing 10% fetal bovine serum as bone marrow lung cells at 5-1° x 1.
Suspend the cells to a total volume of 0.04 cells (10.1 ml) and seed in 0.1 ml portions in a 96-well microplate (Costar).

On the 1st day, 0.1 ml of hyboxanthin, aminopterin, and thymidine (1-(A i' ) medium) was added to each well, and half of the volume was then replaced with HAT medium. Growth of hybrid cells was observed in some wells from around day 5 [1], and hybrid cells stopped growing in almost all wells after 2'iA.

゛ Cloning of raw silk Take 0.1 ml of the culture supernatant from a well where hybrid cells are grown, and incubate it with human 'r' NF fixed on an immunoassay plate (Titertic).
When searching for something that binds this, we were unable to find any wells secreting antibodies capable of binding to human TNF with a probability of approximately 40%.

Only a portion of the cells with high binding ability were selected, and cloning was performed by a limiting dilution method in which cells were planted in a 96-well microplate at a ratio of 1 cell/well.

Some of the obtained clones, 93, were selected and 10
9% in RPMI-1640 medium containing fetal bovine serum.
The culture was scaled up in order of 6-well plate → 24-well plate → 6-well plate → 25 CD flask, and the culture supernatant was collected.

Each supernatant was mixed with 1000 units/ml human '[' NF solution and incubated for 1 hour at 37°C, then L
-929 cells were used to evaluate TNF activity.
11 clones (9C
4G5.8E6B6,1087E11.11D7G4゜8E6C6,2 [32LT10. ')C4A9.8E6
D7.1G7D3.8E6G4, 1087C6) was selected.

Next, the cell culture of the 11 clones was further scaled up using RPM11640 medium containing 10% fetal bovine serum, and 500 ml to 10! Kneaded culture supernatant was collected. This culture supernatant was stirred with 50% saturated ammonium sulfate at 4° C. for about 1 hour, and then centrifuged at 10,0 OOXG for 30 minutes. The pellet was dissolved in a small volume of pure water and dialyzed against 0.1M phosphate buffer pl+8.0.

Add this solution to Protein A Sepharose (Pharmacia).
pH 5.0 or pl+3.0
The monoclonal antibody adsorbed with 0.1M citrate buffer was eluted, neutralized with NaOH, concentrated using a membrane filter (Amicon YM~10), and concentrated to 0.1M citrate buffer.
A purified monoclonal antibody solution was obtained by substituting M phosphate buffer pt18.0.

Example 2 (Inhibition of Schwartzmann reaction by anti-TNF antibody) A male Japanese white heron (approximately 2.5 kg, Kitanan Labes) was used as an experimental animal. In addition, as entodoxy [, co
li 055BS (Difco) was used and dissolved in physiological saline. As a preparatory injection, after trimming the rabbit's flank body hair, give 250μ! It was administered intradermally. There are 1μf and 2μg of endotoxin in it.

4 μg contained 8 μg. Twenty-one hours after the preparatory injection, the drug of Composition Example 3 containing the 11D 7 G 4 antibody obtained in Example 1 as an active ingredient was administered. The dose was 5 μ in terms of antibody amount.

Two hours after antibody administration, i.e., 24 hours after the preparatory injection, endotoxin was administered as a challenge injection at 10 doses per rabbit body weight.
It was administered through the ear vein at a rate of 0 μg.

Five hours after the induction injection of endotoxin, a preparatory injection was performed and the local reactions were observed. Six reactions were scored according to the following preparation.

<-): No change.

(±)　　　: Only more redness occurs.

(10) Causes redness and hyperemia.

(+-1-): Causes petechiae.

(+Shushi): Causes extensive bleeding.

Show the results in the table.

Comparative Example 11 Non-specific mouse IgG instead of D7G4 antibody
The same procedure as in Example 2 was carried out except that (Sigma) was used.

Show the results in the table.

Table: A stronger reaction was observed when the preparation injection was 8 μg than when the injection was 4 μg.

(f) Effects of the Invention By administering the drug of the present invention, it becomes possible to suppress and alleviate systemic shock, local organ disease, and skin JIM disease caused by the Schwartzmann reaction. It is effective as a preventive or therapeutic agent.

Patent applicant Teijin Kaisha Ltd.

Claims (2)

[Claims] (1) Tumor necrosis factor
A Schwartzmann reaction inhibitor whose active ingredient is a substance that has the activity of nullifying TNF (hereinafter referred to as TNF) or removing it from the circulatory system. (2) The Schwartzmann reaction inhibitor according to claim 1, which contains an antibody having specificity for TNF as an active ingredient.