JPH10212230A - Synthesis inhibitor containing dihydroxynaphthoquinone compound of protein belonging to 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: A dihydroxynaphthoquinone compound represented by the following general formula (I) is contained as an active ingredient. Embedded image (Wherein, ^Ra is an amino acid residue whose α-amino group may be optionally protected, or a hydrogen atom)

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a dihydroxynaphthoquinone compound as an active ingredient having a molecular weight of 5 or less.
The present invention relates to an inhibitor for the synthesis of a protein belonging to the heat shock protein group between 7 kilodalton (kD) and 68 kD (hereinafter, referred to as HSP60 family). The inhibitor for the synthesis of a protein belonging to the HSP60 family according to the present invention is particularly an autoimmune disease which is thought to inhibit the synthesis of a protein belonging to the HSP60 family in a tissue, whereby the protein belonging to the HSP60 family is involved in the onset. For example, it is possible to effectively improve the physiological condition of a patient having a disease such as type I diabetes or rheumatoid arthritis, and to effectively treat an autoimmune disease such as type I diabetes or 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, one of the dihydroxynaphthoquinone compounds represented by the formula (I), which is a component of sicon, specifically suppresses the synthesis of a protein belonging to the HSP60 family in cells of a tissue exhibiting a disease state. I found to do. That is, by administering the dihydroxynaphthoquinone compound represented by the formula (I), the synthesis of proteins belonging to the HSP60 family in cells is suppressed, and therefore, the treatment of autoimmune diseases such as type I diabetes and rheumatoid arthritis is suppressed. He found that treatment was possible. The present invention is based on these findings,
An object of the present invention is 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 compound of formula (I):

Embedded image [Wherein, R ^a is ^{a group} represented by the formula (a):

Embedded image Or a hydrogen atom, and R ^b is a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, or a hydroxyl group, a carboxyl group, a carbamoyl group, a mercapto group, or a methylthio group. , Amino group, guanidino group,
Imidazolyl group, phenyl group, hydroxyphenyl group,
Indolyl group, formula (b):

Embedded image And a group represented by the formula (c):

Embedded image Is a linear or branched alkyl group having 1 to 4 carbon atoms, which is substituted by one or more groups selected from the group consisting of groups represented by R ^c and R ^c are each independently Is a hydrogen atom or an amino protecting group;
^b and R ^c are taken together to be a trimethylene group or a trimethylene group substituted with a hydroxyl group, and R ^d is a hydrogen atom or an amino-protecting group]. A molecular weight of from 57 kilodaltons to 6
A heat shock protein synthesis inhibitor of up to 8 kilodaltons.

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 the dihydroxynaphthoquinone compound represented by the formula (I) as an active ingredient. The amino acid residue represented by the formula (a) includes L
Both body and D-forms are included. The amino acid residue represented by the formula (a) is preferably alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine,
Glutamine, lysine, histidine, arginine, aspartic acid, glutamic acid, hydroxylysine, hydroxyproline, norleucine, thyroxine, hydroxyglutamic acid, or ornithine, or derived from these amino acids whose α-amino group is protected with an amino protecting group And more preferably protein constituent amino acids such as alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, glutamine, lysine, histidine, arginine , Aspartic acid, glutamic acid, hydroxylysine, or hydroxyproline, or those in which the α-amino group is protected with an amino protecting group And particularly preferably hydrophobic amino acids, such as alanine, valine, leucine, isoleucine, methionine, phenylalanine, or tryptophan, or those whose α-amino group is protected with an amino protecting group. It can be derived from amino acids. The amino protecting group is not particularly limited, a known amino protecting group,
For example, a benzyloxycarbonyl group, a t-butoxycarbonyl group, a phthalimide group, or the like can be given. R ^a in the dihydroxynaphthoquinone compound represented by the formula (I) is a group represented by the formula (a);
That is, it can be an amino acid residue except for the hydroxyl group of the α-carboxyl group of an amino acid, or a hydrogen atom, and is preferably a hydrogen atom.

The dihydroxynaphthoquinone compound represented by the formula (I) includes various stereoisomers, and any of these pure stereoisomers or a mixture thereof can be used as an active ingredient of the synthesis inhibitor of the present invention. Can be used as an ingredient,
Formula (II):

Embedded image (Wherein R ^a has the same meaning as described above), and preferably a stereoisomer represented by the formula (III):

Embedded image Shikonin represented by:
(R) -5,8-dihydroxy-2- (1-hydroxy-4-methyl-3-pentenyl) -1,4-naphthalenedione; (+)-5,8-dihydroxy-2- (1-hydroxy- 4-Methyl-3-pentenyl) -1,4-naphthoquinone is particularly preferred. Shikonin is contained in, for example, crude drugs such as sicon.

The stereoisomers other than shikonin in the dihydroxynaphthoquinone compound represented by the formula (I) include those represented by the formula (IV):

Embedded image , Ie, (S) -5,8-dihydroxy-2- (1-hydroxy-4-methyl-3-pentenyl) -1,4-naphthalenedione; (-)-5 8-dihydroxy-2- (1
-Hydroxy-4-methyl-3-pentenyl) -1,4
-Naphthoquinone, or shikalkin, which is a racemic form of shikonin and alkanine, and the like.

A derivative which can be easily converted in vivo into the dihydroxynaphthoquinone compound represented by the formula (I), ie, a prodrug, can also be used as the active ingredient of the present invention. Methods commonly used for the selection and manufacture of suitable prodrugs are described, for example, in "Design of Prodrugs" (edited by H. Vanguard, Elsevier, 1985: "Design of Prod").
rugs ", ed. H. Bundgaard, Else
Vier, 1985).

The dihydroxynaphthoquinone compound represented by the above formula (I) contained in the synthesis inhibitor of the present invention is:
It can be prepared by chemical synthesis or by extraction and purification from natural products. Alternatively, a commercially available product may be used. When the dihydroxynaphthoquinone compound represented by the formula (I) used as an active ingredient in the synthesis inhibitor of the present invention is extracted from a natural product, for example, it contains the dihydroxynaphthoquinone compound represented by the formula (I) Whole or part of the plant (eg, whole plant, leaf, root, rhizome, stem, root bark, flower, or fruit) to be processed as it is or simply processed (eg, dried, cut, blanched, steam heated, Or powdered) (for example, a crude drug). The extraction conditions are not particularly limited as long as they are generally used for plant extraction. Examples of the plant containing the dihydroxynaphthoquinone compound represented by the formula (I), in particular, shikonin, are not limited thereto, but include, for example, purple (Lithospe)
rum erythrorhizon Siebol
d et Zuccarini) or Arnebia euchroma (Ro
yl) Johnst. ] Etc. can be used.

In the present invention, when the dihydroxynaphthoquinone compound represented by the formula (I), particularly shikonin, is extracted from a crude drug, the extraction is not limited to this, but it is preferable to extract it from, for example, sicon. Shikon (purple root; Lithospermiradix; Lithos)
The term "permum root" means the root of purple, and these portions can be used alone or in any combination.

The sicon extract which can be used as an active ingredient in the synthesis inhibitor according to the present invention only needs to contain the dihydroxynaphthoquinone compound represented by the above formula (I), in particular, siconin. Crude extracts can be used. The method for producing a silicon extract that can be used in the present invention can be obtained by extracting silicon with water (for example, cold, hot, 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 (eg, 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, and the like. Can be used alone or in an appropriate combination, mixed at a constant ratio, and further used in an anhydrous or water-containing state. Preferably,
Ether and / or dichloromethane and the like 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, (dry)
It is desirable to heat and reflux at a temperature not higher than the boiling point of the silicon or 1 to 300 parts by weight of water or an organic solvent with respect to 1 part by weight of silicon, or to carry out ultrasonic extraction or cold immersion at room temperature. The extraction step is usually performed for 5 minutes to 7 days, preferably 1 minute.
The extraction is performed for 0 minute to 60 hours, and the extraction time can be reduced by adding auxiliary means such as stirring as needed.

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 shikonin extracted and fractionated from a natural product is used in the synthesis inhibitor of the present invention, the crude extract may be used without purification. In addition to the water extract or organic solvent extract by a conventional method, a purified extract obtained by further treating the crude extract with various organic solvents or adsorbents,
It can be used as an active ingredient of the synthesis inhibitor of the present invention. These crude extracts and sicon extracts including purified extracts after various purification treatments may be used in the form of an extracted solution, an extract in which the solvent is concentrated, 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 thus obtained siconium extract contains the shikonin contained in the sicon and simultaneously the impurities derived from the sicon as the raw material.

The synthesis inhibitor of the present invention comprises a dihydroxynaphthoquinone compound represented by the above formula (I) or an extract of a plant containing the dihydroxynaphthoquinone compound represented by the above formula (I). The extract of a crude drug containing the dihydroxynaphthoquinone compound represented by I) (particularly, sicon extract) is used alone or, preferably, together with a pharmaceutically or veterinarily acceptable common carrier. , Animals, preferably mammals (especially humans). As a dosage form,
There is no particular limitation, for example, powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts, or oral preparations such as pills, or injections,
Parenteral preparations such as liquids for external use, ointments, suppositories, creams for topical administration, or eye drops can be mentioned. These oral preparations 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, stearic acid. Excipients such as magnesium, polyethylene glycol, magnesium silicate, anhydrous silicic acid, or synthetic aluminum silicate, binders, disintegrants, surfactants, lubricants, glidants, diluents, preservatives, coloring Using agents, flavors, flavors, stabilizers, humectants, preservatives, or antioxidants,
It can be manufactured according to a conventional method. For example, capsules filled with a mixture of 1 part by weight of shikonin and 99 parts by weight of lactose are filled.

Examples of parenteral administration include injection (subcutaneous, intravenous, etc.) and rectal administration. Of these, injections are most preferably used. For example, in the preparation of an injection, a dihydroxynaphthoquinone compound represented by the formula (I) as an active ingredient or a plant extract containing the dihydroxynaphthoquinone compound represented by the formula (I), for example, In addition to a crude drug extract (particularly, a sicon extract) containing the dihydroxynaphthoquinone compound represented by the formula (I), for example, a water-soluble solvent such as physiological saline or Ringer's solution, a vegetable oil or a fatty acid ester or the like A water-soluble solvent, an isotonic agent such as glucose or sodium chloride, a solubilizer, a stabilizer, a preservative, a suspending agent, an emulsifier, or the like can be optionally used. 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.

Although the synthesis inhibitor of the present invention is not limited to this, the dihydroxynaphthoquinone compound represented by the formula (I) may be present in an amount of 0.01 to 99% by weight, preferably 0.1 to 80% by weight. It can be contained in an amount of% by weight.
In addition, an extract of a plant containing the dihydroxynaphthoquinone compound represented by the formula (I), for example, an extract of a crude drug containing the dihydroxynaphthoquinone compound represented by the formula (I) (particularly, sicon extract ) As an active ingredient may be prepared by appropriately adjusting the amount of the dihydroxynaphthoquinone compound represented by the formula (I) contained therein to fall within the above range. it can. In addition, a plant extract containing the dihydroxynaphthoquinone compound represented by the formula (I),
For example, when a synthetic inhibitor containing, as an active ingredient, an extract of a crude drug containing the dihydroxynaphthoquinone compound represented by the formula (I) (particularly, sicon extract) is used as a preparation for oral administration, It is preferable to formulate using a pharmaceutically acceptable carrier. The dosage in the case of using the synthetic inhibitor of the present invention varies depending on the type of disease, age, sex, weight, degree of symptoms, degree of symptoms, administration method and the like, and is not particularly limited. The amount of the dihydroxynaphthoquinone compound to be administered is usually orally or parenterally, about 1 mg to 10 g per adult, divided about 1 to 4 times a day. 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.

[0026]

As described above, the dihydroxynaphthoquinone compound represented by the formula (I) contained in the synthesis inhibitor of the present invention has an effect of specifically inhibiting the synthesis of a protein belonging to the HSP60 family in cells. Therefore, when the dihydroxynaphthoquinone compound represented by the formula (I) is administered, the biosynthesis of a protein belonging to the HSP60 family in cells is specifically reduced. Accordingly, the dihydroxynaphthoquinone compound represented by the formula (I) is
A protein belonging to the HSP60 family can be used for the prevention and treatment of autoimmune diseases related to its onset, such as type I diabetes and rheumatoid arthritis.
That is, 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 a dihydroxynaphthoquinone compound represented by the formula (I) as an active ingredient. Also concerns. The present invention also provides a therapeutic agent for an autoimmune disease, comprising as an active ingredient a plant extract containing the dihydroxynaphthoquinone compound represented by the formula (I).
For example, it relates to a therapeutic agent for type I diabetes or a therapeutic agent for rheumatoid arthritis. 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 an extract of sicon as an active ingredient.

[0027]

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) Shikonin treatment and heat shock treatment Two days after seeding, shikonin (Ichimaru Falcos) represented by the above formula (III) was added to the medium of the gastric cancer cell line KATO III so as to have a final concentration of 1 μM. 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 performed in the same manner as described above except that no shikonin was added.

(3) Measurement of HSP expression level in cultured human cancer cells Each cell treated in the above section (2) was 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 protein quantification was performed, 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 shikonin 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 shikonin was added, the concentration of the band having a molecular weight of about 60 kD was significantly reduced as compared with the control test. That is, it can be concluded that shikonin has the activity of a synthetic inhibitor that suppresses the expression of HSP60.

[0034]

As described in detail above, the dihydroxynaphthoquinone compound represented by the above formula (I) can
It has the activity of a synthetic inhibitor that suppresses the expression of proteins belonging to the SP60 family. Therefore, by administering the dihydroxynaphthoquinone compound represented by the formula (I), for example, 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 obtained. The physiological condition can be effectively improved, and the disease can be effectively treated.

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Claims (3)

[Claims] 1. A compound of formula (I): [Wherein R ^a is ^{a group} represented by the formula (a): Or a hydrogen atom, and R ^b is a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, or a hydroxyl group, a carboxyl group, a carbamoyl group, a mercapto group, or a methylthio group. , Amino group, guanidino group,
Imidazolyl group, phenyl group, hydroxyphenyl group,
Indolyl group, formula (b): And a group represented by the formula (c): Is a linear or branched alkyl group having 1 to 4 carbon atoms, which is substituted by one or more groups selected from the group consisting of groups represented by R ^c and R ^c are each independently Is a hydrogen atom or an amino protecting group;
^b and R ^c are taken together to be a trimethylene group or a trimethylene group substituted with a hydroxyl group, and R ^d is a hydrogen atom or an amino-protecting group]. A molecular weight of from 57 kilodaltons to 6
Inhibitor of heat shock protein synthesis up to 8 kilodaltons. 2. Formula (I): [Wherein, R ^a is ^{a group} represented by the formula (a): Or a hydrogen atom, and R ^b is a hydrogen atom, a linear or branched alkyl group having 1 to 4 carbon atoms, or a hydroxyl group, a carboxyl group, a carbamoyl group, a mercapto group, or a methylthio group. , Amino group, guanidino group,
Imidazolyl group, phenyl group, hydroxyphenyl group,
Indolyl group, formula (b): And a group represented by the formula (c): Is a linear or branched alkyl group having 1 to 4 carbon atoms, which is substituted by one or more groups selected from the group consisting of groups represented by R ^c and R ^c are each independently Is a hydrogen atom or an amino protecting group;
^b and R ^c are taken together to be a trimethylene group or a trimethylene group substituted with a hydroxyl group, and R ^d is a hydrogen atom or an amino-protecting group]. An inhibitor of synthesis of a heat shock protein having a molecular weight of from 57 kDa to 68 kDa, comprising a plant extract as an active ingredient. 3. A molecular weight of from 57 kDa to 68 kDa, characterized by containing an extract of sicon as an active ingredient.
Inhibitor of heat shock protein synthesis up to kilodaltons.