JPH02223529A - Remedy comprising cystatin as active ingredient - Google Patents

Abstract

PURPOSE:To obtain an allergic agent effective for treating bone diseases such as osteoporosis and allergic dermatosis, having effectively inhibitory action on liberation of calcium from bone, comprising cystatin as an active ingredient. CONSTITUTION:A remedy comprising cystatin as an active ingredient. Cystatin is protein existing in animal cell and body fluid. Cystatin strongly inhibits protein hydrolyzing activity of cysteine protease and has possibility of a remedy for various diseases caused by extraordinary increase in cysteine protease. The antiallergic agent can be parenterally or orally administered by intravenous or intra-arterial medication in both cases of bone and allergic diseases.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a therapeutic agent containing cystatin as an active ingredient,
More specifically, the present invention relates to an antiallergic agent and a bone disease therapeutic agent containing cystatin as an active ingredient.

[Prior Art] Cystatin is a protein present in animal cells and body fluids, and is a type of cysteine protease inhibitor. Today, various cystatins have been isolated and their structures determined, and they act within cells.
, cystatin β, etc. Cystatin C is known to act by being secreted outside the cell.

That is, in 1982, Grubb et al. isolated the first cystatin, cystatin C, from brain cerebrospinal fluid and determined its structure [Proc, Natl, Hecad, Sci, U, S
, 3024 (1982)], then Katsuura et al.
em, Biophys, Res, Commun,, 1
15, 902 (1983)], cystatin β was isolated and structure determined from the liver in 1984 [Bioch
em.

Biophys, Res, Commun, , 121
, 149 (1984) Since 1° cystatin strongly inhibits the proteolytic activity of cysteine protease, its potential as a therapeutic agent for various diseases caused by an abnormal increase in cysteine 10-tease is being studied. For example, since cysteine protease derived from the virus is involved in the replication process of poliovirus in host cells, and cystatin inhibits this cysteine protease, the possibility of cystatin as an agent for suppressing viral infection is being investigated. [Proc, Na
Mouth Acad, Sci, tl, S, ^,, 83.5392
(1986)].

In addition, an abnormal increase in cysteine propase is ISi! It has also been suggested that it could be used as a therapeutic agent for muscular atrophic diseases [Metabolism, 24 (2), Seeing with the Eye, Page 275 (1)].
987)].

[Problems to be Solved by the Invention] An object of the present invention is to provide a new medical use of cystatin, that is, a new use of cystatin as an antiallergic agent and a therapeutic agent for bone diseases.

[Means for Solving the Problem 1] As a result of intensive research aimed at developing new medicinal uses for cystatin, the present inventor found that cystatin significantly suppresses the release of calcium from bones in pathological models such as osteoporosis. It also strongly suppressed IOE antibody production in mice. Therefore, we discovered that cystatin is extremely effective as a therapeutic agent for bone diseases and as an antiallergic agent.
The invention has been completed.

That is, the present invention provides an antiallergic agent containing cystatin as an active ingredient, and a bone disease therapeutic agent containing cystatin as an active ingredient.

Cystatin used in the present invention may be either an intracellular type that acts within cells or an extracellular type that is secreted outside the cell and acts. Specifically, cystatin α (derived from rats), cystatin Δ (derived from humans), cystatin β (derived from rats),
Intracellular cystatins such as cystatin B (human origin);
and cystatin C (human origin), cystatin S (human origin)
Examples of extracellular cystatin include cystatin EW (derived from egg white), cystatin colosterum (derived from human), and cystatin colosterum (derived from human). Among these, human-derived cystatins such as cystatin A1 cystatin B1 cystatin C are preferred. Cystatin C is one of the particularly preferred ICs in the present invention because it has the property of being easily taken up into cells. These cystatins can be isolated from animal cells or body fluids such as serum and saliva [Proc, Natl, Ac
ad, Sci, tl, S, A, 79, 3024
(1982); Biochem, Bioph vs.
ResCommun, 115.902 (1983)
;BiochemBiophys, Res, Con+
mun, 121, 149 (1984);
Protein Inhibitors, l), 1
25, Jpn, 5cSoc, Prcss and
Springer-Verlag (1983)
:Cysteine proteinases a
ndTheir Inhibitorsl), 497
, Walter de Gruyter &
Co., Berlin (1986): F.E.
BS Lett, 186.41 (1985)].

Moreover, these cystatins may be recombinant cystatins produced by common genetic engineering techniques. Recombinant cystatin can be obtained, for example, by chemically or biochemically synthesizing the genes encoding cystatin α and cystatin A, inserting them into appropriate plasmids, and expressing them in Escherichia coli. Examples include recombinant cystatin α (Japanese patent application No. 62-21706) and recombinant cystatin A (Japanese patent application No. 63-110402).

The present inventors have revealed that cystatin significantly inhibits the release of calcium from bones in pathological models such as osteoporosis, and also strongly inhibits IOE antibody production in mice. Therefore, cystatin is effective in the treatment of bone diseases such as osteoporosis, Behget's disease, hypercalcemia, and osteomalacia, and also in the treatment of allergic diseases such as allergic skin diseases, allergic inflammation, and allergic purpura. It is effective for

Cystatin can be administered intravenously, intraarterially, intramuscularly, nasally, subcutaneously, or in cases of bone or allergic diseases.
Administration can be by parenteral administration, such as intrarectally, or by oral administration.

Preparations for parenteral administration such as intravenous, intraarterial, intramuscular, and subcutaneous administration include injections and infusions, and these preparations can be prepared by methods known per se.

Injections, infusions, and the like can be prepared by lyophilizing cystatin alone or with a carrier such as mannyl, lactose, albumin, etc. and filling it into vials. It may also be a ribosome preparation consisting of a thin film of phospholipid such as lecithin, lysolecithin, or sphingomyelin. It may also be an implant-type preparation that is placed subcutaneously.

Examples of formulations for nasal administration include powders comprising cystatin and carriers such as cell L1-su lower alkyl ethers such as hydroxypropyl cellulose, polyacrylates such as sodium polyacrylate, and crystalline cellulose. .

Preparations for rectal administration include suppositories.

Suppositories are prepared by combining the commonly used suppository base with cacao butter and surfactants such as fatty acid esters, polyoxyethylene derivatives, pyridoxine fatty acid esters, sucrose fatty acid esters, and phospholipids. There are suppositories that use one or more active agents and are uniformly dispersed in the above-mentioned base to improve absorption efficiency.

Preparations for oral administration include the above-mentioned IC liposome preparations.

When cystatin is used for allergic diseases, it can also be administered as a spray that is sprayed into the oral cavity or nose.

All of the above-mentioned preparations can be prepared by methods known per se, and commonly used preservatives, coloring agents, stabilizers, etc. can be added to these preparations as necessary.

The dosage of cystatin may vary depending on the patient's age, weight, symptoms, and the type of cystatin administered, but
For bone disease treatment, usually 0.1 to 500 try/K
y body weight/day, preferably 10-1001115 F body weight/day
days, and in the case of allergic disease treatment, it is usually 0.1 days.
~5001FJ/9 body weight/[1, preferably 10-2
9 body weight/day in 0011r'j/.

[Effects of the Invention] The therapeutic agent for bone diseases of the present invention containing cystatin as an active ingredient is extremely effective in treating osteoporosis, Behcet's disease, hypercalcemia, etc., and is also an antiallergic agent containing cystatin as an active ingredient. is extremely effective in treating allergic skin diseases, allergic rhinitis, etc.

[Example] The present invention will be specifically described below with reference to Test Examples and Examples.

Test Example 1 For bone calcium release, 5-week-old 11-star male rats were fed a low-calcium diet (
About 0.02%, Oriental Yeast Co., Ltd. standard A modified blend sample) was raised for one week and used for experiments.

Cystatin α (Recombinant cystatin α is produced by chemically and biochemically synthesizing the gene encoding cystatin α, inserting it into a plasmid, and transforming E. coli to express it in E. coli. 2 5 n+H, pH isotonic with NaCl.

7.5 in Tris-HCI buffer, administration container 0
．． 6. The mice were administered via the tail vein at a ratio of d/100g body weight.

Blood was collected 3 hours after administration of cystatin α, and calcium concentration in the serum was measured using the OCPC method [H, J, Git
leman: Anal, Biochem, , Vol.
18.521 (1967)].

The results are shown in Table 1.

Table 1 As shown in Table 1, calcium release from bones was significantly suppressed by administration of cystatin α601RI/Kg body weight.

Test Example 2 A group of 7 to 10 8-week-old BDF1 female mice were used for the test. Cystatin α, similar to that used in Test Example 1 and used in 1c, was dissolved in phosphate buffer-physiological saline to serve as the test drug, and a drug containing no active ingredient was administered intraperitoneally to separate groups of mice as a control. .

Aluminum hydroxide 4 adsorbed 1 μg of ovalbumin
ml was intraperitoneally administered for sensitization, and at the same time the test drug was intraperitoneally administered. On the 14th day after sensitization (day 14), blood was collected from the mouse orbital vein, diluted 2 times with physiological saline, centrifuged, and the obtained serum was used as an antiserum against the primary sensitization.

In addition, on the 25th day after sensitization (day 25), the same amount of antigen was administered intraperitoneally as a booster, and on the 41st day after sensitization (da
V41), blood was collected in the same manner, and the obtained serum was used as an antiserum for secondary sensitization.

Using the obtained antiserum, InternaAr
Chives of^llcrgy and Appl
ied Immunolooy,.

Vol 48.16 (1975) (Ovary,
Z, Ca1azzaS, S, and s, ojim
a: PCA reactions with mouse
antibodies in m1ce and r
Passive cutaneous anaphylaxis (PCA) described in
) The amount of antibody produced was determined by the reaction.

That is, the collected serum was diluted to various concentrations and added to different rats (
A female Wistar (male, weight 200-250 g) was injected. After 4 hours, add 4 ml of ovalbumin to 0.5
% Evans blue physiological saline solution, this was administered intravenously, and the serum boundary concentration of dye exudation was determined. The results are shown in Table 2.

As a result, it is clear that J: lycystadine α suppresses the production of ICIE.

Test Example 3 Effect of IE on Cystatin in Mice Eight-week-old BDF1-type mice were used in the test in a group of eight mice. Cystatin A was dissolved in physiological saline to serve as a test drug, and physiological saline containing no active ingredient was used as a control.

Four tays of nofluminium hydroxide adsorbed with 1 μg of ovalbumin was administered intraperitoneally to sensitize the mice, and at the same time the test drug was administered intravenously. On day 14 after sensitization, blood was collected from the orbital vein of the mouse, diluted 2 times with physiological saline, and centrifuged to obtain antiserum. The obtained antiserum was used in Test Example 2 to determine the amount of ICIE antibody produced by a PCA reaction similar to that of IC. The results are shown in Table 3. As is clear from the results, cystatin A significantly suppressed the production of IQE antibodies.

Test Example 4 Toxicity Test Even when 1 g of the same cystatin α used in Test Example 1 was administered intraperitoneally or intravenously per 14 mice, no changes were observed in the mice.

Example 1 Injection 600g of lyophilized cystatin α and 6d of physiological saline for injection
was added and dissolved, and after sterile filtration (0.22 nm caliber, manufactured by Nippon Millipore Industries, Ltd.), the two precepts were filled into an ampoule, and the ampoule space was replaced with nitrogen gas and sealed to obtain an injection.

Example 2 All egg yolk lecithin 110μmol, cholesterol 50μ
Dissolve mol in chloroform solution of 10-
After mixing thoroughly, the solvent was distilled off under reduced pressure in a 40°C hot bath to form a thin film on the inner wall of the flask.

It was further vacuum dried in a desiccator for 3 hours. 30 μmol of cystatin Δ was dissolved in 10 d of physiological saline, placed in the above-mentioned flask, shaken with a voldexing mixer, subjected to ultrasonic waves, and then passed through a filter (manufactured by Millibore 7) with a diameter of 0.22 nllll. . Ten volumes of this solution were filled into an ampoule, and the ampoule space was replaced with nitrogen gas and sealed to obtain a liposome preparation for injection.

Example 3 Suppositories 1.0 q of sodium glycolate was added to 17 g of cacao fat and mixed thoroughly in a mortar. Cystatin α2 was collected in a mortar, and the two bases were gradually added thereto and mixed to form a homogeneous single-dose composition, which was filled into a predetermined container 29 times to obtain a crude drug preparation.

Example 4 Powder for Nasal Administration 100 mg of lyophilized cystatin A and hydroxylzolopylcellulose were collected in a mortar and mixed thoroughly to form a uniform powder composition. A cystatin preparation for nasal administration was obtained by filling this composition into a predetermined capsule.

Claims (2)

[Claims] (1) Anti-allergic agent containing cystatin as an active ingredient. (2) A therapeutic agent for bone diseases containing cystatin as an active ingredient.