JPS6351335A - Degranulation inhibitor for mast cell - Google Patents

Abstract

PURPOSE:To obtain the titled inhibitor containing a Bowman-Birk type trypsin inhibitor derived from soybean as an active ingredient. CONSTITUTION:A degranulation inhibitor for mast cells obtained by containing a Bowman-Birk type trypsin inhibitor derived from soybean (BBI; 6,000-8,000 mol.wt., present in water-soluble fraction of soybean together with a Kunitz type trypsin inhibitor having about 20,000mol.wt.) as an active ingredient. The natural BBI is obtained by concentration and fractionation using extraction with an aqueous medium or polar organic solvent, e.g. lower alcohol, lower aliphatic ketone, etc., separation with a membrane, isoelectic precipitation, etc., to give a crude purified substance, subjecting the resultant substance to purifying means, e.g. gel filtration, etc. Since the BBI is capable of acting on mast cells and inhibiting the degranulation thereof, it may be effective against allergic diseases.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to chymase and tributase inhibitors, particularly mast cell degranulation inhibitors.

(Prior art) rgE (immunoglobulin E
It has already been clarified that mast cells in one tissue play an important role in allergic inflammation associated with

(Refer to June 1978, published by Igaku Shoin, 111). That is, it is thought that intracellular granules are released by antigen binding to IgH bound to the cell surface, and histamine, 5RS-A, etc. contained therein cause inflammation.

On the other hand, Katsunuma et al. found that quibuze (Ghy) was present in 70 mast cells.
It was revealed that this enzyme exists and is essential for mast cell degranulation, and that the free enzyme degrades IgG and creates neutrophil migration factor (Seikagaku, Vol. 57, p. 1076). Furthermore, Katsunuma et al. revealed that degranulation was suppressed by antibodies against chymase and chymostatin (C1lBOstaLin), which has a chymase inhibitory effect, but aprotinin (aprotinin)
in) and α1-antichymotrypsin (α1-anti
In protein chymase inhibitors with molecular Q8000 or higher, such as chymotrypsin, the inhibition was hardly observed ("Biochemistr71nt," 10.pp.
, 883-871 (1985)) [Problems to be Solved by the Invention] During further research, the inventors discovered that tryptase (Tryptase), which exists in mast cell granules in addition to chymase,
Ase) is prothrombia (Prothra-m
Obtaining the knowledge that it is possible to activate thrombin (bin) into thrombin, and that the possibility of involvement in inflammatory reactions via the blood coagulation system due to this activation is important,
Based on this and the experimental facts regarding chymase mentioned above, we have come to realize that inhibition of two types of proteases in mast cell granules, namely chymase and tryptase, may be meaningful in suppressing degranulation and, in turn, suppressing allergic reactions. However, to date, no polymeric inhibitor has been found that exhibits strong activity against both proteases, particularly against chymase.

However, if such a high-molecular-weight inhibitor were to be discovered, it would be difficult to indiscriminately invade cells other than the target mast cells through midline diffusion, as is the case with other low-molecular-weight inhibitors. It is assumed that this will open up prospects for the development of safe drugs without the fear of side effects.

[Process of Completion of the Invention] Therefore, the present inventors have developed a plant protease inhibitor, which has not been known to have basic physiological behavior, although the existence of various polymeric protease inhibitors has been known for some time. As I continued my research, I discovered BowIlan Birk, which is derived from soybeans.
)-type trypsin inhibitor has a significant inhibitory effect on both enzymes, and it has been found that it effectively suppresses IgE-induced histamine release (11) from mast cell granules.

[Means for solving the problem] Unexploited No. 11 is based on the above knowledge, and its gist is that soybean-derived Bowman-Bark (Bowman B)
irk) type trypsin inhibitor as an active ingredient.

Here, the soybean-derived Bowman-Birk trypsin inhibitor (hereinafter abbreviated as BBI), which is the main subject of the invention, has a molecular weight of about 6,000 to 8,000, while other types of trypsin inhibitors have a molecular weight of about 2. Kunit
z) p) Present in the water-soluble fraction of soybean together with lipunin inhibitor.

Incidentally, the basic properties of these two types of inhibitors are rMeL
hod in EnzymologyJ Volume 19 853
(1970) and “New Perspectives on White Matter Research (Focusing on Chemical Research)” published by Kyoritsu Shuppan (1982), and their structures have already been determined (Eur.
,J,Bio-ches, ,32.417;J,Bi
ochem, 74, 8S7 (1973)) Its synthesis and modification by chemical and biochemical methods is also possible today. Therefore, BBI as used herein is a concept that encompasses not only the natural Bowman-Birk type trypsin inhibitor originally obtained from soybean, but also its active fragments or modifications, or chemical or biochemical compounds thereof.

By the way, various methods are already known for preparing natural BBI. It can be made by any method, but in general, soybeans, defatted soybeans, soybean whey, etc. are used as starting materials, and this is mixed in an aqueous medium or a polar organic solvent (e.g., lower alcohols or lower). After first preliminarily purifying the composition to a crudely purified state by extraction with a group of ketones or dioxane, membrane separation, isoelectric precipitation, concentration by salting out, and fractionation, this composition is Furthermore, by applying purification methods such as gel filtration, ion exchange, or adsorption, it is possible to purify the specimen to a specimen that shows a single pattern in polyacrylamide gel disk electrophoresis. A crude product is sufficient as long as it is clear.The activity can be assayed using known methods such as the Kunitz casein digestion method or synthetic substrates (e.g. N-Benzoylargi).
nine-p-'n1toroanilide) can be used. In addition? SDS-containing polyacrylamide gel electrophoresis or high-performance liquid chromatography may be used to test for purity and purity.

[Effect] BBI derived from soybean strongly inhibits chymase and tryptase purified from mast cells. This inhibitory mechanism is
It is understood that this is due to the specific binding action to the active centers of chymotrypsin and trypsin enzymes in the molecular chain of BBI.

Furthermore, soybean-derived BBI inhibits degranulation from mast cells. This effect is thought to be the result of an inhibitory effect on both of the enzymes mentioned above, but considering the molecular weight of BBI (approximately 8.0 QQ), it is unlikely that some uptake mechanism exists at the interface with the cell membrane during this time. It would be certain.

However, due to its high molecular nature, the BBI mentioned above is not considered to invade into cells of other species other than the target cell by simple diffusion, as is the case with other low-molecular-weight inhibitors, and in fact, it is believed that the above BBI does not invade into cells of other species other than the target cell by simple diffusion, as it does with other low-molecular-weight inhibitors. No effects on cells other than cells have been discovered so far.

As described above, since BBI acts on mast cells and suppresses their degranulation, it is expected to be effective as a powerful drug for allergic diseases.

[Examples] Hereinafter, the invention will be described in more detail with reference to Examples and Reference Examples, but the examples are of course for illustration only and do not mean a limitation on the idea of the invention.

Reference Example 1 (Preparation example of BBI) Soybean whey obtained in the process of producing isolated soybean protein from low-denatured defatted soybeans was concentrated, and this C linear activity (crude protein fund 75
．． 5%) Add 0.5 volumes of acetone to 1 volume and stir for about 1 hour, then centrifuge and separate.
of acetone and stirred for about 1 hour, the resulting precipitate kj,
The fractions were dialyzed against wood. To this dialysate was added 150 volumes of 0.5 M sodium phosphate buffer (p) 17. After adjusting the pH to 7.0 by adding Q), it was passed through a DEAE-cellulose ion exchange column and absorbed into the resin.
The column was eluted with a linear salt gradient from 0 to 0.4 M;
The eluate was fractionated using an aracin collector, and BBI
Two fractions were obtained, one enriched with Kunitz-type trypsin inhibitor and the other enriched with Kunitz-type trypsin inhibitor.

The BBI-rich fraction is e-nishiki, and after dialysis, its pH is adjusted to 4.0.
was prepared, passed through a CM cellulose ion exchange column, and the adsorbed BBI was eluted with a 0-015M salt concentration gradient, and the eluate was lyophilized to obtain purified BBI. Both parts of the Kunitz mold were also freeze-dried after isoelectric precipitation to obtain purified inhibitors. The purity of each sample was determined by SO8-containing polyacrylamide gel electrophoresis and high performance liquid chromatography (
When measured by gel filtration method, both proteins contained 9.
It was 5% or more. Also, the specific activity of BBI is.

Sigma trypsin “Type-XIJ (7500
~9000 BAEE units/mg of active protein)
and synthetic substrate (N-Benzyl-DL-Argini
When measured using ne-p-nitroanil ide: BAPA), it was 5 to 6 units/g protein (
The inhibitory activity was defined as fun when the activity of trypsin to thaw 2 units of BAPA under the same conditions was inhibited by 50%.

).

Reference Example 2 (Inhibition of protease by BBI) The inhibitory ability for chymase purified from rat tongue and tryptase purified from rat intraperitoneal mast cells was determined by adding BBI and Kunitz-type inhibitor, and in the case of chymase, S
ue-Leu-Val-Tyr-MCA (Sue = succinyl, MCA = 4-methylcoumaryl-7-amide) as substrate, measured at p) 18.5, and in the case of tributase, Boc-Phe-Ser- Ar
g-MGA (Boc=tertiary butyroxycarbonyl) was measured at pH 7.5 as A (quality).BBI
Aqueous solution of and Norikotsutu type inhibitor or Kymostachiy
(C:bymosLaLin: (Foundation) Protein Research! Nikokai) dimethyl sulfoxide solution was added to chymase or tryptase, and after incubating at 25°C for 5 minutes,
Substrate solution was added. The enzyme reaction purified product was measured using a fluorometer to determine the 50% inhibition rate of each 511 harmful agent. The results are shown in Table 1 below.

Table 1 As is clear from Table 1 above, the inhibitory ability of Kunitz-type inhibitors against chymase was not strong, and the inhibitory ability of chymostatin against tryptase was also weak. BB
Only I is strong against both chymase and tryptase3
11 showed harmful effects. This is a characteristic that is not seen in conventional inhibitors.Example (Suppression of degranulation by BBI)
Mast cells with a purity of 95 to 99% were prepared from the peritoneal cavities of rats with elevated IgE antibody levels after subcutaneous inoculation of gy1usbraciliensis larvae.

Degranulation was measured using free histamic acid as an indicator.

i.e. 4 x 105 mast cells per 11%
Suspended in Tyrode's solution containing bovine serum albumin, BB
I or other inhibitors for 0 to 60 minutes 3
An IgE antibody was added, and the amount of histamine released within 10 minutes at 37°C was determined by liquid chromatography. The control without inhibitor was 100.
The results expressed as % are shown in Table 2.

(Leaving space below) Table 2 As is clear from the above table, the low molecular weight inhibitors chymostatin and leupeptin showed the effect of suppressing histamine release, and the effect of the former was particularly remarkable. On the other hand, the macromolecular inhibitors α1-antigimotrypsin and aprotinin have no effect of inhibiting histamine release, although they themselves have a chymase inhibitory effect, and the latter in particular promoted histamine IS.

In contrast, although BBI is a polymeric inhibitor, it showed the effect of effectively suppressing the release of histamine.

[Effects of the Invention] As explained above, soybean-derived BBI strongly inhibits chymase and tryptase in mast cells and suppresses degranulation from these cells, so it is effective against inflammation and other allergic symptoms. Adaptation is expected.

Claims (1)

[Claims] (1) Bowman-Bark (Bowman B) derived from soybean
A mast cell degranulation inhibitor containing a trypsin inhibitor (irk) type trypsin inhibitor as an active ingredient.