JPH10120567A - Aconitine-containing synthetic inhibitors of proteins belonging to the HSP60 family - Google Patents

Abstract

(57) Abstract: An autoimmune disease in which a heat shock protein (HSP60 family) having a molecular weight of 57 kDa to 68 kDa is involved in the onset thereof (for example, I
Provided is an agent for inhibiting the synthesis of a protein belonging to the HSP60 family, which is capable of effectively improving the physiological condition of a patient with type 2 diabetes and rheumatoid arthritis, and effectively treating the disease. SOLUTION: It contains aconitine or a stereoisomer thereof as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an aconitine as an active ingredient having a molecular weight of 57 kilodalton (k).
The present invention relates to inhibitors of the synthesis of proteins belonging to the group of heat shock proteins between D) and 68 kD (hereinafter referred to as HSP60 family). HSP60 according to the invention
Synthetic inhibitors of proteins belonging to the family, in particular,
By suppressing the in-tissue synthesis of proteins belonging to the HSP60 family, proteins belonging to the HSP60 family are thought to be involved in the pathogenesis of autoimmune diseases, for example, patients with diseases such as type I diabetes and rheumatoid arthritis. It can effectively improve physiological conditions and effectively treat autoimmune diseases such as type I diabetes and rheumatoid arthritis.

[0002]

2. Description of the Related Art In recent years, autoimmune diseases have become a major problem. An autoimmune disease is a disease in which the immune system, which normally should not attack the components that make up your body, reacts with your own tissue and destroys it.
Includes type I diabetes and rheumatoid arthritis. For example, in recent years in Japan, with the development of economy, society, and culture, as well as the improvement of living standards and changes in lifestyles, the number of diabetic patients has increased significantly, and the condition has become more severe and complicated. Advances in diabetology have improved patient prognosis, but have increased frequent retinopathies, nephropathy, and neuropathy, as well as accelerated arterial stiffness, which has severely impaired health and social activities . Among diabetes, type I diabetes (insulin-dependent diabetes mellitus; IDD)
The incidence of M) has increased several fold in many countries in the last few decades, with 1% of currently living humans becoming I by the age of 70.
It is expected that type 2 diabetes will occur.

[0003] Type I diabetes is a disease in which only β cells of the pancreatic islets of Langerhans, which are insulin-producing cells, are autoimmunely destroyed, resulting in an insulin deficiency state, and is an organ-specific autoimmune disease (Atkinson, MA et al .:
"Sci. Am.", 263 : 42-49, 1990; Todd JA .: "Immuno
l. Today ", 11 : 122-129, 1990. The autoimmune process that causes type I diabetes is very strictly limited to the pancreas, and often begins before adulthood.
When type I diabetes develops clinically, there is pancreatic islet inflammation (isletitis), and the majority of the insulin-producing beta cells are specifically lost (Atkinson, MA, et al .: "
Sci. Am. ", 263 : 62-67, 1990). The clinical manifestations of diabetes only appear after the majority (perhaps more than 90%) of the beta cells have been destroyed to the extent that they cannot be regenerated, and the survival of the patient depends on insulin. Will depend on the external supply of
By the time it can be detected by clinical diagnosis, type I diabetes has many problems, including that this autoimmune response has already irreversibly damaged many of them without significant subjective symptoms. I have.

[0004] Rheumatoid arthritis is a chronic inflammatory disease in which the synovium of the joint is the main lesion. The site of the lesion is sometimes not limited to the synovium of the joint, and it is not uncommon for it to extend to the whole body. The inflammation that initially occurs in the synovium of the joint eventually causes synovial proliferation, further destruction of cartilage and bone, and destruction of joint tissue. As a result, the patient is not only severely restricted socially and at home, but also has a considerable financial burden. The number of patients with rheumatoid arthritis accounts for 0.1-0.3% of the population. This is an example of confirming rheumatoid arthritis, and it is expected that the number of patients will increase to about 10 times that of rheumatoid arthritis, including suspected cases and rheumatoid arthritis-related diseases.

On the other hand, heat shock proteins (heat shock proteins)
protein; also called HSP, stress protein)
Cells with some stress, such as heat, heavy metals, drugs,
A group of proteins expressed in cells by stimulation with amino acid analogs or hypoxia (low oxygen concentration). Heat shock proteins are ubiquitous in nature and are produced by higher animals, including bacteria, yeast, plants, insects, and humans.

[0006] HSPs are of various types,
Due to the molecular weight, the HSP90 family (for example, 9
0 kD or 110 kD HSP, etc.), HSP70 family (eg, 70-73 kD HSP, etc.), HS
P60 family (eg, 57-68 kD HSP etc.), small molecule HSP family (eg, 20 kD, 2
5-28 kD or 47 kD HSP). In the present specification, an HSP having a specific molecular weight is indicated by an HSP and a numeral described immediately after the HSP. For example, an HSP having a molecular weight of 60 kD is referred to as “HSP60”. As described above, there are many types of HSPs, which differ not only in molecular weight but also in structure, function, or property. In addition to the response to stress, some of these proteins are constitutively synthesized and, under normal circumstances, regulate protein folding, unfolding, protein subunit assembly, and protein membrane trafficking. It has been shown to play an essential physiological role. These functions as heat shock proteins are called molecular chaperones.

[0007] One of the attentions regarding the etiology of autoimmune diseases is molecular homology. In other words, when a self-antigen has a common antigenicity with a foreign antigen such as a microorganism, antibodies and sensitized lymphocytes produced by microbial infection attack their own tissues by cross-reaction, resulting in the development of an autoimmune disease. (Atkinson, MA et al .: "Sci. A
m. ", 263 : 42-49, 1990; Shinha, AA et al .:" Scie
nce ", 248 : 1380-1388, 1990). For example, bacterial HSP
Proteins belonging to 60 families include tuberculosis, leprosy,
It is a major antigen such as syphilis, veterans' disease, or Lyme disease (Young, RA et al .: "Cell", 59 : 5-8, 1989),
In addition, proteins belonging to the HSP60 family of bacteria have strong immunogenicity and have molecular homology with their own (host human) proteins, so that they are not affected by infectious disease-triggered molecular homology. It is believed that autoimmune diseases develop.

For example, an antibody (64 kD) that reacts with a 64 kD protein detected in the blood of diabetic patients and their families
D autoantibodies) are β-cell specific, and often appear immediately before diabetes is diagnosed. That is, pancreatic islet cell antigen, which is considered to be the cause of onset in type I diabetes, is a glycoprotein having a molecular weight of 64 kD (Baekkeskov, S. e.
t al .: "Nature", 298 : 167-169, 1982). 64
Antibodies against the kD protein are not limited to human diabetes (Atkinson, MA et al .: "Lancet", 335 : 1357-136).
0, 1990), BB rats (Baekkeskov, S. et al .: "Scie
nce ", 224 : 1348-1350, 1984) and NOD mice (Atkins
on, MA et al .: "Diabetes", 37 : 1587-1590, 1988)
As described above, it is also detected in a type I diabetes model animal that naturally develops type I diabetes and exhibits many characteristics of human type I diabetes. The 64 kD protein of pancreatic β-cell in type I diabetes may be a heat shock protein because it is induced by cytokines and heat stimulation.

The 64 kD protein of the pancreatic Langerhans islet β-cell in NOD mouse, a model animal of type I diabetes, is derived from H. tuberculosis (Mycobacterium tuberculosis).
It has been shown to be an autoantigen that is immunologically cross-reactive with antibodies to proteins belonging to the SP60 family. As described above, the immunological crossover between the protein belonging to the HSP60 family and the 64 kD protein autoantigen was observed.
D protein may be a member of the HSP60 family, suggesting that autoimmune mechanisms that cross epitopes of proteins belonging to the HSP60 family are involved in the development of type I diabetes. When a T lymphocyte clone having specificity for a protein belonging to the HSP60 family of Mycobacterium tuberculosis is transferred, it causes Langerhans insulitis and hyperglycemia in young NOD mice. In addition, when a protein belonging to the HSP60 family of Mycobacterium tuberculosis is injected into NOD mice by an immunogenic administration method, that is, by injection into a NOD mouse together with an adjuvant, diabetes can be developed early (Elias, D. et al .: "Proc. Natl. Acad. . Sci.
USA ", 87 : 1576-1580, 1990). HS of mycobacteria
These facts that the immune response of animals to proteins belonging to the P60 family cause type I diabetes are due to the attack by the immune system on antigens that cross-react with antibodies to proteins belonging to the HSP60 family of mycobacteria, damaging β cells. It is shown that.

It is known that proteins belonging to the HSP60 family are related to rat adjuvant arthritis, which is an animal model for rheumatoid arthritis, and to human rheumatoid arthritis. For example, in the case of rheumatoid arthritis, among the heat shock proteins that are bacterial cell proteins, proteins belonging to the HSP60 family have been found to have molecular homology to proteoglycans present in articular cartilage. The involvement of protein-reactive T lymphocytes belonging to the HSP60 family has been shown in adjuvant arthritis in rats ("C
urr. Top. Microbiol. Immunol. ", 145 : 27-83, 198.
9). The disease is transferred to irradiated immunologically naive rats by transferring a clone of T lymphocytes reactive to proteins belonging to the HSP60 family of M. tuberculosis. ("Science", 219 : 56-5
8, 1983; "Nature", 331 : 171-173, 1988). The T lymphocytes also show cross-reactivity with joint proteoglycans ("Proc. Natl. Acad. Sci. USA", 82 : 5117-512).
0, 1985). Regulatory T lymphocytes induced by proteins belonging to the HSP60 family are also recognized in arthritis due to streptococcus and pristine. Thus, adjuvant arthritis appears to be an autoimmune disease caused by protein T lymphocytes belonging to the anti-HSP60 family. Also, in human juvenile rheumatoid arthritis, HS
The involvement of protein-reactive T lymphocytes belonging to the P60 family is considered.

In addition, T lymphocytes that specifically react with proteins belonging to the HSP60 family derived from mycobacteria have been extracted from the synovial fluid of patients with rheumatoid arthritis ("Lancet", II : 478-). 480, 1988; "Na
ture ", 339 : 226, 1989;" Annu. Rev. Immunol. ", 1
1 : 637, 1993). Thus, the mycobacterial HS
Proteins that show cross-reactivity with proteins belonging to the P60 family are found at high concentrations in the cartilage / pannus junction of rheumatoid arthritis, whereas they are found in normal tissues and tissues that exhibit chronic inflammation due to other diseases. No ("Scand. J. Immunol.", 31 : 283-288, 1990). Furthermore, antibodies against proteins belonging to the HSP60 family are detected in human and rat rheumatoid arthritis (Kaufmann, SHE, et al .: "Immunol. Today", 11 :
129-136, 1990), it is possible that the etiology of rheumatoid arthritis is autoimmunity against self antigens similar in structure to proteins belonging to the HSP60 family of mycobacteria. Thus, the presence of an immune response to proteins belonging to the HSP60 family is associated with both rat and human arthritis.

[0012]

SUMMARY OF THE INVENTION In view of the above circumstances, the present inventors have been able to effectively improve the physiological condition of patients with autoimmune diseases such as type I diabetes and rheumatoid arthritis, In order to develop a method that can effectively treat immune diseases, various studies have been made on compounds that exhibit a synthesis inhibitory effect on proteins belonging to the HSP60 family. As a result, we have:
Surprisingly, the present inventors have found that aconitine, a component of Bushi, specifically suppresses the synthesis of proteins belonging to the HSP60 family in cells of a tissue exhibiting a pathological condition. That is, by administering aconitine, intracellular H
It has been found that the synthesis of proteins belonging to the SP60 family is suppressed, and thus it is possible to treat autoimmune diseases such as type I diabetes and rheumatoid arthritis.
The present invention is based on such findings, and has as its object to provide a synthesis inhibitor of a protein belonging to the HSP60 family, which can effectively treat autoimmune diseases such as type I diabetes and rheumatoid arthritis. .

[0013]

Accordingly, the present invention provides a heat shock protein having a molecular weight of from 57 kilodaltons to 68 kilodaltons, which comprises aconitine or a stereoisomer thereof as an active ingredient. ,
HSP60 family protein).

As used herein, the term "HSP60 family" refers to a molecule having a molecular weight of 57 kD to 68 kD, as described above.
Mean heat shock proteins. In addition, HSP6
Examples of proteins belonging to family 0 include H
SP60 (ie, a heat shock protein with a molecular weight of 60 kD), HSP58 (ie, a heat shock protein with a molecular weight of 58 kD), HSP65 (ie, a heat shock protein with a molecular weight of 65 kD), or GroEL
(Ie, a prokaryote, for example, a heat shock protein having a molecular weight of about 64 kD, such as E. coli).

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The synthesis inhibitor of the present invention contains aconitine or a stereoisomer thereof as an active ingredient. Aconitine which can be used as an active ingredient in the synthesis inhibitor of the present invention has the formula (I):

Embedded image And contained in, for example, crude drugs such as bush. Aconitine has stereoisomers, and any pure stereoisomer or a mixture thereof can be used as an active ingredient of the synthesis inhibitor of the present invention.

Aconitine contained in the synthetic inhibitor of the present invention can be prepared by chemical synthesis or by extracting and purifying from natural products. Or,
A commercially available product may be used. When aconitine used as an active ingredient in the synthetic inhibitor of the present invention is extracted from a natural product, for example, whole or a part of a plant containing aconitine (for example, whole plant, leaf, root, rhizome, stem, root bark) ,
Extraction is performed using the flower (or fruit) as it is or by using a simple processing (eg, drying, cutting, blanching, steam heating, or powdering) (eg, a crude drug). The extraction conditions are not particularly limited as long as they are generally used for plant extraction. Examples of plants containing aconitine include, but are not limited to, Aconitum carmichael (Aconitum carmichael)
i Debeaux, Aconit (Aconit)
um japonicum Thumb. ), Aconitum brachypod
oduum Diels) or Aconitum kusnezofiii Re
ichb. ) Etc. can be used.

When aconitine is extracted from a crude drug in the present invention, it is not limited to this, but it is preferable to extract aconitine from, for example, bush. Acon
Iti tuber (Aconite root) refers to tuberous root, mountain aconite, or a tuberous root of a congener plant, and these parts can be used alone or in any combination.

The bush extract which can be used as an active ingredient in the synthesis inhibitor according to the present invention only needs to contain the above-mentioned aconitine, and thus a crude bush extract can be used. The method for producing a bush extract that can be used in the present invention can be obtained by extracting bushes with water (for example, cold water, hot water, or hot water), or by extracting with an organic solvent. .
As the organic solvent, for example, alcohol (for example, methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol, or butyl alcohol), ester (for example, methyl acetate, ethyl acetate,
Propyl acetate or butyl acetate), ketone (eg, acetone or methyl isobutyl ketone), ether, petroleum ether, n-hexane, cyclohexane,
Toluene, benzene, a halogen derivative of a hydrocarbon (eg, carbon tetrachloride, dichloromethane, or chloroform), pyridine, glycol (eg, propylene glycol or butylene glycol), polyethylene glycol, or acetonitrile can be used. Organic solvents can be used alone or in appropriate combination, mixed at a certain ratio, and further used in an anhydrous or water-containing state. Preferably, methyl alcohol and / or chloroform are desirable. As a method of water extraction or organic solvent extraction, a method used for ordinary crude drug extraction can be used. For example, for 1 part by weight of (dried) bush,
It is desirable to heat and reflux at a temperature not higher than the boiling point of the mixture or to carry out ultrasonic extraction or cold immersion at a temperature lower than the boiling point while stirring with water or an organic solvent in an amount of 3 to 300 parts by weight. The extraction step is usually performed for 5 minutes to 7 days, preferably for 10 minutes to 60 hours, and the extraction time can be shortened by adding auxiliary means such as stirring, if necessary.

After completion of the extraction step, a crude extract can be obtained by separating insolubles from the water or organic solvent extract by a suitable method such as filtration or centrifugation. When aconitine extracted and fractionated from a natural product is used in the synthesis inhibitor of the present invention, the crude extract may be used as it is without any particular purification. In addition to the water extract or organic solvent extract by a conventional method, a purified extract obtained by further treating the above crude extract with various organic solvents or adsorbents is also used as an active ingredient of the synthesis inhibitor of the present invention. Can be. The crude extract and the bush extract containing the purified extract after various purification treatments may be used as is, or may be an extract obtained by concentrating the solvent, or the solvent may be distilled off. A dried powder, a crystallized and purified substance, or a viscous substance may be used, or a diluent thereof may be used. The bush extract thus obtained contains aconitine contained in the bush and at the same time contains impurities derived from the raw material bush.

The synthetic inhibitor of the present invention comprises aconitine or an extract of a plant containing aconitine, for example, an extract of a crude drug containing aconitine (particularly, a bush extract).
It can be administered to animals, preferably mammals (especially humans), alone or preferably together with conventional pharmaceutically or veterinarily acceptable carriers. The dosage form is not particularly limited. For example, oral preparations such as powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts, or pills, or injections And parenteral agents such as topical solutions, topical solutions, ointments, suppositories, creams for topical administration, and eye drops. These oral agents include, for example, gelatin,
Sodium alginate, starch, corn starch, sucrose,
Lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soy lecithin, sucrose, fatty acid esters, talc, magnesium stearate, polyethylene glycol, magnesium silicate, anhydrous silicic acid, synthetic aluminum silicate, etc. Excipients, binders, disintegrants, surfactants, lubricants, glidants, diluents, preservatives, coloring agents,
It can be produced according to a conventional method using a flavor, a flavoring agent, a stabilizer, a humectant, a preservative, an antioxidant and the like.
For example, a capsule filled with 1 part by weight of aconitine and 99 parts by weight of lactose is filled.

Examples of parenteral administration methods include injection (subcutaneous, intravenous, etc.) and rectal administration. Of these, injections are most preferably used. For example, in the preparation of an injection, in addition to aconitine as an active ingredient or an extract of a plant containing aconitine, for example, an extract of a crude drug containing aconitine (particularly, a bush extract), for example, Water-soluble solvents such as saline or Ringer's solution, water-insoluble solvents such as vegetable oils or fatty acid esters, isotonic agents such as glucose or sodium chloride, solubilizers, stabilizers, preservatives, suspending agents, and emulsifiers Etc. can be used arbitrarily. Further, the synthetic inhibitor of the present invention may be administered using a sustained-release preparation technique using a sustained-release polymer or the like. For example, the synthetic inhibitors of the present invention can be incorporated into a pellet of ethylene vinyl acetate polymer and the pellet can be surgically implanted into the tissue to be treated.

The synthesis inhibitor of the present invention contains, but is not limited to, aconitine in an amount of 0.01 to 0.99% by weight, preferably 0.1 to 0.80% by weight. be able to. In addition, the synthetic inhibitor of the present invention containing an aconitine-containing plant extract, for example, an aconitine-containing crude drug extract (particularly, a bush extract) as an active ingredient, contains aconitine contained therein. It can be prepared by appropriately adjusting the amount to fall within the above range. In addition, when a plant extract containing aconitine, for example, a synthetic inhibitor containing an aconitine-containing crude drug extract (particularly, a bush extract) as an active ingredient, is used as a formulation for oral administration, It is preferable to formulate using a pharmaceutically acceptable carrier. The dose when using the synthetic inhibitor of the present invention depends on the type of disease,
Although it depends on the age, sex, weight, degree of symptoms, administration method, etc. of the patient, there is no particular limitation, but the amount of aconitine is usually about 1 mg to 100 mg per adult per day.
It is orally or parenterally administered in about 4 times. Furthermore, the use is not limited to pharmaceuticals, and various uses, for example, functional foods and health foods can be given in the form of food and drink.

[0023]

As described above, aconitine contained in the synthesis inhibitor of the present invention has an action of specifically inhibiting the synthesis of proteins belonging to the HSP60 family in cells. Biosynthesis of proteins belonging to the HSP60 family is specifically reduced. Therefore, the aconitine can be used for the prevention and treatment of autoimmune diseases in which proteins belonging to the HSP60 family are related to its onset, such as type I diabetes and rheumatoid arthritis. That is, the present invention also relates to a therapeutic agent for an autoimmune disease, for example, a therapeutic agent for type I diabetes or a therapeutic agent for rheumatoid arthritis, which comprises aconitine or a stereoisomer thereof as an active ingredient. Further, the present invention provides a therapeutic agent for an autoimmune disease, for example, a therapeutic agent for type I diabetes or a therapeutic agent for rheumatoid arthritis, comprising as an active ingredient an extract of a plant containing aconitine or a stereoisomer thereof. Related to Furthermore, the present invention also relates to a therapeutic agent for an autoimmune disease, for example, a therapeutic agent for type I diabetes or a rheumatoid arthritis, which comprises a bush extract as an active ingredient.

[0024]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention. Example 1: Measurement of HSP expression level in cultured human cancer cells (1) Culture of cultured human cancer cells Gastric cancer cell line KATO III (ATCC HTB 10
3) RPMI16 containing 10% inactivated fetal bovine serum
The cells were cultured in a 40 medium at 37 ° C. under 5% carbon dioxide except for the heat shock treatment.

(2) Aconitine treatment and heat shock treatment Two days after seeding, aconitine represented by the above formula (I) (Matsuura Pharmaceutical Co., Ltd.) was added to the medium of the gastric cancer cell line KATO III at a final concentration of 100 μM. And cultured for 24 hours.
Thereafter, the cells were subjected to a heat shock treatment at 45 ° C. for 15 minutes, and then cultured at 37 ° C. overnight. The control test was carried out as described above, except that no aconitine was added.

(3) Measurement of HSP expression level in cultured human cancer cells The cells treated in the above (2) were homogenized by the following method, and the HSP expression level was measured by Western blotting. That is, the cells treated in the above (2) were treated with phosphate buffered saline [composition: KCl = 0.2 g /
1, KH ₂ PO ₄ = 0.2 g / l, NaCl = 8 g /
1, Na ₂ HPO ₄ (anhydrous) = 1.15 g / l;
PBS (-)), and then lysed buffer (lysis buffer) [1.0% NP-4
0, 0.15 M sodium chloride, 50 mM Tris-HC
1 (pH 8.0), 1 mM of 5 mM-EDTA, 2 mM-N-ethylmaleimide, 2 mM phenylmethylsulfonyl fluoride, 2 μg / ml leupeptin and 2 μg / ml pepstatin], and allowed to stand on ice for 20 minutes.
Thereafter, centrifugation was performed at 12,000 rpm at 4 ° C. for 20 minutes. 10 μl of the supernatant after centrifugation was added to PBS (−) 790
μl, add protein assay stain (Dye Reag
ent Concentrate: Bio-Rad, Catalog No. 500-00
06) 200 μl was added. After standing at room temperature for 5 minutes, the protein was quantified by measuring the absorbance at 595 nm.

Using the sample for which the protein was quantified, L
aemmli buffer system (Laemmli, NK, "Natur
e ", 283 : pp. 249-256, 1970), lysates containing equal amounts of protein were subjected to SDS polyacrylamide gel electrophoresis. After electrophoresis, blotting and subsequent blocking were performed. Using a transfer device (Trans-Blot Electrophoretic Transfer Cell: Bio-Rad, Catalog No. 170-3946), adhere the gel to a 0.45 μm nitrocellulose membrane (Schleicher & Schuell, Catalog No. 401196) at room temperature and 100 V at room temperature. A tris glycine buffer (Tris Gly Running and Bloom) containing 0.025 M Tris and 0.192 M glycine and adjusted to pH 8.5 was used as a blotting buffer.
tting Buffer; Enprotech, Massachusetts, USA
A buffer prepared by adding methyl alcohol to catalog number SA100034) to 20% was used. After blotting, the nitrocellulose membrane was added to a 10% skim milk (Snow Brand Milk Products) -PBS (-) solution at room temperature for 30 minutes.
Incubate to block non-specific binding.

After blocking, an anti-human HSP60 mouse monoclonal antibody (Stre
ssGen, Victoria, BC, Canada, Cat.No. SPA-8
06), a primary antibody reaction was performed. This anti-human HSP
60 murine monoclonal antibody is an antibody to human HSP60 prepared was prepared as an immunogen by recombinant DNA methods using E. coli ( "J. Exp. Med." 175
, 1805-1810, 1992), mammalian HSP60 (primate HS)
P60, mouse HSP60, rat HSP60, and hamster HSP60) ("J. Exp. M
ed. " 175 , 1805-1810, 1992). This anti-human HSP60
The epitope recognized by the mouse monoclonal antibody is located in the amino acid sequence consisting of amino acid residues 383 to 447 of the human HSP60 amino acid sequence ("J. Exp. Med." 175 , 1805-1810, 1992). ). After the primary antibody reaction, the solution was replaced with PBS (-) for 5 minutes, and the solution was washed twice with a slow rocking shaker. Further, PBS (-)-0.1% Tween20 was added.
(Bio-Rad, Cat. No. 170-6531) 15 in solution
The solution was replaced every minute for four washes. Finally, the cells were washed twice with PBS (-) for 5 minutes each.

After the washing was completed, a peroxidase-labeled goat anti-mouse IgG antibody (CAPPEL, Catalog No. 55550) was
% For 2 hours using 5 ml of an antibody solution prepared by diluting 5000-fold with a PBS (-) solution containing 5% skim milk.
A secondary antibody reaction was performed. After the completion of the reaction, the nitrocellulose membrane was washed twice with a PBS (-) solution for 5 minutes while changing the solution twice, and further with a PBS (-)-0.1% Tween20 solution for 15 minutes for 5 times. Performed by slow rocking shaker. Finally PBS (-)
The solution was washed twice for 5 minutes each. Extra PBS
(-) After removing the solution, the western blotting detection reagent (ACL Western blotting detection reagent; Ame
rsham, catalog number RPN2106), sprinkle onto the nitrocellulose membrane, incubate for 1 minute, remove excess detection reagent, wrap the nitrocellulose membrane in wrap, and cover the reaction surface with X-ray film (Kodak X-OMAT, AR, (Catalog No. 165 1454), exposed, developed and HSP6
The existence of 0 was examined.

As a result, in the control test, that is, in the gastric cancer cell line KATO III to which aconitine was not added, one band having a molecular weight of about 60 kD was detected. The molecular weight was determined by binding to the anti-human HSP60 mouse monoclonal antibody and molecular weight markers (egg white ovalbumin and bovine serum albumin). In the gastric cancer cell line KATO III to which aconitine was added, the concentration of the band having a molecular weight of about 60 kD was significantly lower than that in the control test. That is, it can be concluded that aconitine has the activity of a synthetic inhibitor that suppresses the expression of HSP60.

[0031]

As described in detail above, aconitine has the activity of a synthetic inhibitor that suppresses the expression of proteins belonging to the HSP60 family in cells. Therefore, by administering aconitine, for example, the physiological condition of a patient with an autoimmune disease (eg, type I diabetes, rheumatoid arthritis, etc.) associated with the onset of a protein belonging to the HSP60 family can be effectively improved, Can be treated effectively.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07M 9:00

Claims (3)

[Claims] 1. A heat shock protein synthesis inhibitor having a molecular weight of from 57 kDa to 68 kDa, comprising aconitine or a stereoisomer thereof as an active ingredient. 2. A heat shock protein synthesis inhibitor having a molecular weight of from 57 kDa to 68 kDa, comprising as an active ingredient a plant extract containing aconitine or a stereoisomer thereof. 3. A heat shock protein synthesis inhibitor having a molecular weight of from 57 kDa to 68 kDa, comprising an extract of Bushi as an active ingredient.