JP5587637B2 - Matrix metalloprotease inhibitors and uses thereof - Google Patents

Description

  The present invention relates to matrix metalloprotease inhibitors, and more specifically glycosaminoglycans such as hyaluronic acid oligosaccharides and keratan sulfate oligosaccharides screened for properties on MMP-9 and MMP-12 activity. The invention relates to use as a matrix metalloproteinase inhibitor and an elastase activity inhibitor, and a therapeutic agent for chronic obstructive pulmonary disease.

First, abbreviations used in the application documents will be described.
MMP: matrix metalloproteinase COPD: chronic obstructive pulmonary disease HA: hyaluronic acid CS: chondroitin sulfate DS: dermatan sulfate GAG: glycosaminoglycan GPC: gel permeation chromatography HA4: hyaluronic acid tetrasaccharide (GlcA β1-3 GlcNAc β1- 4 GlcA β1-3 GlcNAc)
L4: Keratan sulfate disaccharide (Gal6S β1-4 GlcNAc6S)
L4L4: Keratan sulfate tetrasaccharide (Gal6S β1-4 GlcNAc6S β1-3 Gal6S β1-4 GlcNAc6S)

  Various cells constituting animal tissues are adhered and fixed by various extracellular matrix molecules. MMP, which is also deeply involved in cell proliferation, differentiation, migration, and apoptosis, is a generic name for a group of metallophilic proteases that use such extracellular matrix protein as a main substrate. MMPs are currently MMP-1, MMP-3, gelatinase A (MMP-2), neutrophil collagenase (MMP-18), gelatinase B (MMP-9), macrophage elastase (MMP-12), collagenase ( Dozens of types such as MMP-13) are known. These common properties include having zinc in the active domain, and adjusting the activity by a natural inhibitor (Tissue Inhibitor of Metalloproteinase) specific to MMP present in the living body. (Non-Patent Document 1).

  In addition, as a result of studies by the present inventors, gene expression levels of MMP-9, 12, and 13 were significantly increased in Fut8 knockout mice (COPD spontaneous model mice), which are related to alveolar destruction. Has been reported (Non-patent Document 2). From this fact, it is considered effective to inhibit the MMP activity that acts in the final stage in order to prevent alveolar destruction. However, various MMP inhibitors have been developed so far, but none of them has improved therapeutic effects due to serious side effects.

  COPD is a disease group that occurs frequently in middle-aged and smokers and presents chronic and persistent obstructive ventilatory impairment, and is ranked fourth in the cause of death by World Health Organization statistics. In Japan, COPD is defined as a disease characterized by obstructive ventilation disorder caused by pulmonary emphysema, chronic bronchitis, or a combination of the two according to the Japanese Respiratory Society Guidelines (Non-Patent Document 3). Of these, emphysema is estimated to be about 3 million patients and reserves, including those with mild illness.

  The pathophysiology of emphysema is characterized by the destruction of the airways and alveolar walls below the terminal bronchioles and irreversible airway dilatation, with no pathological fibrosis and obstruction of the airway system. Is defined. Regarding the mechanism of the development of emphysema, the “protease / antiprotease imbalance theory”, apoptosis of alveolar walls (Non-patent Documents 4 and 5), and the like have been considered, but many have not yet been clarified. The most certain intrinsic risk factor is α1-antitrypsin deficiency, which is a genetic disease, but it is very rare in Japan (Non-patent Document 3).

  Regarding the action of GAG on MMP, it has been reported that proteoglycan mainly composed of chondroitin sulfate C (chondroitin 6 sulfate) having a molecular weight of 500 kDa or more derived from an aqueous extract of cartilage fish cartilage inhibits MMP. (Patent Document 1). As for polysulfated GAG, for example, the influence on MMP activity has been studied for polysulfated CS. However, polysulfated CS has been reported to inhibit MMP-3 but activate MMP-1, indicating that polysulfated CS is not a preferred substance as an MMP inhibitor. (Non-Patent Document 6). In addition, as a drug for suppressing obstructive ventilation disorder, a drug characterized by inhibiting the production or accumulation of chondroitin sulfate proteoglycans in the lung is known (Patent Document 2). Patent Documents 3 and 4 describe that the therapeutic effect of pulmonary fibrosis and interstitial lung disease can be expected as a fibrosis-inhibiting effect of keratan sulfate oligosaccharide and its derivatives. Therefore, it cannot be predicted whether the oligosaccharide of keratan sulfate has a therapeutic effect for lung diseases other than pulmonary fibrosis and interstitial lung disease such as emphysema. Patent Document 5 exemplifies emphysema, chronic obstructive pulmonary disease, and the like as examples of indications for growth factor inducers containing GAG having a molecular weight of about 5,000 to 2,000,000 as an active ingredient.

  However, it has not been known that GAG having a specific structure significantly inhibits MMP activity and that L4 is useful as a therapeutic agent for COPD.

WO2004 / 083257 Japanese Patent No.4101276 JP-A-11-269076 JP 2000-256385 A JP2002-3384

Kaoru Miyazaki, Biochemistry, 68, 1791-1807, 1996 Wang, X et al. Proc. Natl. Acad. Sci. USA 102, 15791-15796, (2005) Respiratory Society COPD Guidelines Preparation Committee: Guidelines for COPD Diagnosis and Treatment 1999 Hoshino Y, Am. J. Physiol. Lung cell Mol. Physiol. 281 (2) p509-516 (2001) Aoshiba K, Am. J. Respir. Crit. Care Med. 153 (2) p530-534 (2003) Andrew Nethery et al., Biochem. Pharmacol., 44, p1549-1553 (1992)

  In view of the above situation, the present invention provides a new means for inhibiting MMP related to GAG, and an object of the present invention is to provide a way to treat chronic obstructive pulmonary disease (COPD) and the like. .

  As a result of intensive studies to solve the above problems, the present inventors have found that GAG having a specific structure has an activity to significantly inhibit MMP, and have completed the present invention.

  That is, the present invention comprises HA oligosaccharide, keratan sulfate oligosaccharide, 6-O-sulfated N-acetylheparosan and N, 6-O-sulfated heparosan, and pharmaceutically acceptable salts thereof. The present invention provides an MMP inhibitor containing at least one selected from the group as an active ingredient (hereinafter referred to as “the MMP inhibitor of the present invention”). In addition, the above-described GAG oligosaccharide, heparosan having a specific structure, and pharmaceutically acceptable salts thereof are hereinafter collectively referred to as “GAG oligosaccharide and the like”.

The MMP inhibitor of the present invention more preferably contains as an active ingredient one or more selected from the group consisting of GAG oligosaccharides represented by the following formulas HA4, L4 and L4L4.
HA4: GlcA β1-3 GlcNAc β1-4 GlcA β1-3 GlcNAc
L4: Gal6S β1-4 GlcNAc6S
L4L4: Gal6S β1-4 GlcNAc6S β1-3 Gal6S β1-4 GlcNAc6S
(In the formula, Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, GlcA represents a glucuronic acid residue, 6S represents a sulfate group at the 6-position, β1-3 represents β1- (3 glycoside bonds, β1-4 represents β1-4 glycoside bonds, respectively)

  The MMP inhibitor of the present invention is particularly preferably used as an application for inhibiting MMP-9, MMP-12 and MMP-13, and more preferably used as an application for inhibiting MMP-9.

The present invention also provides cell-derived MMP activity by at least one or more selected from the group consisting of GAG oligosaccharides represented by the following formulas HA4, L4 and L4L4 and pharmaceutically acceptable salts thereof. It is an invention that provides a method for inhibiting MMP inhibition (hereinafter referred to as “inhibition method of the present invention”).
HA4: GlcA β1-3 GlcNAc β1-4 GlcA β1-3 GlcNAc
L4: Gal6S β1-4 GlcNAc6S
L4L4: Gal6S β1-4 GlcNAc6S β1-3 Gal6S β1-4 GlcNAc6S
(In the formula, Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, GlcA represents a glucuronic acid residue, 6S represents a sulfate group at the 6-position, β1-3 represents β1- (3 glycoside bonds, β1-4 represents β1-4 glycoside bonds, respectively)

  The inhibition method of the present invention is particularly preferably used for inhibiting MMP-9, MMP-12, and MMP-13, and particularly preferably used for inhibiting MMP-9.

  The present invention also provides an elastase activity inhibitor (hereinafter referred to as “the present elastase inhibitor”) containing at least a keratan sulfate oligosaccharide.

The keratan sulfate oligosaccharide that is an active ingredient of the elastase inhibitor of the present invention is preferably a keratan sulfate oligosaccharide represented by the following formula L4.
L4: Gal6S β1-4 GlcNAc6S
(In the formula, Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, 6S represents a sulfate group at the 6-position, and β1-4 represents a β1-4 glycoside bond. )

The present invention also provides a therapeutic agent for chronic obstructive pulmonary disease (hereinafter referred to as “the therapeutic agent for pulmonary disease of the present invention”) comprising a keratan sulfate oligosaccharide represented by the following formula L4.
L4: Gal6S β1-4 GlcNAc6S
(In the formula, Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, 6S represents a sulfate group at the 6-position, and β1-4 represents a β1-4 glycoside bond. )

  The chronic obstructive pulmonary disease is more preferably used when it is emphysema.

  In the present invention, “agent” means “preparation product” or “active ingredient itself”. “Dispensing product” has the same meaning as “composition”, but “agent” is used as a common expression in the pharmaceutical field. Whether it is a “preparation product” or an “active ingredient itself” can be clearly distinguished by the definition of the agent. That is, "agent containing ~ as an active ingredient" means "preparation product", and "agent consisting of" means "active ingredient itself". For example, the MMP inhibitor of the present invention includes “HA oligosaccharide, keratan sulfate oligosaccharide, 6-O-sulfated N-acetylheparosan and N, 6-O-sulfated heparosan, and pharmaceutically acceptable ones thereof. MMP-inhibiting composition containing at least one selected from the group consisting of salts of The same applies to the elastase inhibitor of the present invention and the pulmonary disease therapeutic agent of the present invention, and is the same as the “elastase activity suppressing composition” and the “chronic obstructive pulmonary disease therapeutic composition” containing the respective active ingredients. It is significant. Moreover, the agent which consists of said each active ingredient has shown the use of each active ingredient itself.

In addition, the present invention provides at least one or more selected from the group consisting of GAG oligosaccharides represented by the following formulas HA4, L4, and L4L4 and pharmaceutically acceptable salts thereof, and a test drug. A method for selecting a drug (hereinafter referred to as “the present invention screening method”), which is brought into contact with a cell and screened for a promoting action or a mitigating action on the suppression of MMP activity derived from the cell by the GAG oligosaccharide or salt in the test drug. ).
HA4: GlcA β1-3 GlcNAc β1-4 GlcA β1-3 GlcNAc
L4: Gal6S β1-4 GlcNAc6S
L4L4: Gal6S β1-4 GlcNAc6S β1-3 Gal6S β1-4 GlcNAc6S
(In the formula, Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, GlcA represents a glucuronic acid residue, 6S represents a sulfate group at the 6-position, β1-3 represents β1- (3 glycoside bonds, β1-4 represents β1-4 glycoside bonds, respectively)

  According to the screening method of the present invention, the elastase inhibitory action of the above-mentioned agent of the present invention is promoted and formulated in combination with the agent of the present invention, thereby further enhancing the therapeutic effect on chronic obstructive pulmonary diseases such as emphysema. It is possible to select active ingredients that can be used. On the contrary, the elastase inhibitory action of the agent of the present invention can be alleviated, and for example, the active ingredient of the mitigating agent when the side effects caused by the agent of the present invention become apparent can be selected.

  According to the present invention, there are provided an MMP inhibitor comprising GAG oligosaccharide or the like as an active ingredient, an MMP inhibition method using the same, an elastase activity inhibitor and a therapeutic agent for chronic obstructive pulmonary disease. MMP inhibitors are extremely useful because they are expected to be applicable to various other diseases. Furthermore, the present invention provides a selection method capable of selecting an active ingredient capable of promoting or alleviating the action of the above agent.

It is a figure which shows the influence on MMP activity according to various GAG oligosaccharide groups. It is drawing which showed the result of having examined the degree of MMP-9 suppression by changing the coupling | bonding sugar chain number of HA oligosaccharide. It is a figure which shows the density | concentration dependence of the MMP activity inhibitory effect of keratan sulfate oligosaccharide. It is a figure which shows the inhibition constant with respect to MMP-9 by GM6001. It is a figure which shows the inhibition constant with respect to MMP-9 by keratan sulfate oligosaccharide L4. It is a figure which shows the inhibition constant with respect to MMP-9 by keratan sulfate oligosaccharide L4L4. It is drawing which examined the accumulation inhibitory effect of the total cell number by L4. It is drawing which examined the accumulation inhibitory effect of the neutrophil count by L4. It is drawing which examined the influence on the neutrophil count ratio by L4. It is drawing which examined the inhibitory effect of the elastase induction emphysema by L4. It is drawing which showed the inhibitory effect of the elastase induction emphysema by L4 with the formalin fixed microscope image.

<1> MMP inhibitor of the present invention The MMP inhibitor of the present invention comprises HA oligosaccharide, keratan sulfate oligosaccharide, 6-O-sulfated N-acetylheparosan and N, 6-O-sulfated heparosan, and pharmaceuticals. An MMP inhibitor containing, as an active ingredient, at least one selected from the group consisting of:

[A] Active ingredient The GAG oligosaccharide used in the MMP inhibitor of the present invention may be composed of a single species or a mixture.

  Moreover, each GAG oligosaccharide etc. are suitably adjusted with the method of WO96 / 16973 or WO02 / 04471, for example. GAG used as the raw material can be obtained from proteoglycan or chicken crown of cartilaginous fish such as sharks. Moreover, what is marketed can also be used. Specific methods for producing GAG oligosaccharides and the like are specifically disclosed in the Examples section.

(1) HA oligosaccharide For example, HA obtained by a known means is decomposed into an oligosaccharide by a hyaluronic acid degrading enzyme, and the oligosaccharide is condensed by an ordinary method such as ethanol precipitation, gel filtration and anion exchange chromatography. The HA oligosaccharide used in the present invention can be produced by purification using a graphic purification method or the like. The HA oligosaccharide is preferably the following HA4.

HA4: GlcA β1-3 GlcNAc β1-4 GlcA β1-3 GlcNAc
(In the formula, GlcA represents a glucuronic acid residue, GlcNAc represents an N-acetylglucosamine residue, β1-3 represents a β1-3 glycoside bond, and β1-4 represents a β1-4 glycoside bond.)

(2) Keratan sulfate oligosaccharide As a method for preparing keratan sulfate oligosaccharide, endo-β-N-acetylglucosaminidase-type keratan sulfate degrading enzyme such as keratanase derived from Bacillus bacteria is used in a buffer solution of keratan sulfate or highly sulfated keratan sulfate. It can be obtained by fractionating the resulting degradation product after (II) or degradation by reacting with Keratan sulfate degrading enzyme derived from Bacillus circulans KsT202 strain. The obtained oligosaccharide fraction can be separated and purified from the target keratan sulfate oligosaccharide by using, for example, concentration by ethanol precipitation, gel filtration, and decomposition / purification by anion exchange chromatography. The keratan sulfate is preferably L4 or L4L4 described below.

L4: Gal6S β1-4 GlcNAc6S
L4L4: Gal6S β1-4 GlcNAc6S β1-3 Gal6S β1-4 GlcNAc6S
(In the formula, Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, GlcA represents a glucuronic acid residue, 6S represents a sulfate group at the 6-position, β1-3 represents β1- (3 glycoside bonds, β1-4 represents β1-4 glycoside bonds, respectively)

(3) 6-O-sulfated N-acetylheparosan 6-O-sulfated N-acetylheparosan is prepared by dissolving N-acetylheparosan obtained by a known means in distilled water and then in the presence of a catalyst. , 6-O-sulfation can be performed, and separation and purification can be performed using, for example, concentration by ethanol precipitation, gel filtration, and decomposition / purification by anion exchange chromatography.

(4) N, 6-O-sulfated heparosan N, 6-O-sulfated heparosan is obtained by, for example, subjecting a polysaccharide obtained by known means to hydrazine decomposition and N-deacetylation. The product is N-sulfated, further 6-O-sulfated in the presence of a catalyst, and separated and purified using, for example, concentration by ethanol precipitation, gel filtration, and decomposition / purification by anion exchange chromatography. Can do.

(5) Pharmaceutically acceptable salt The pharmaceutically acceptable salt is a salt of the GAG oligosaccharide of the above (1) to (3), that is, the salt of the HA oligosaccharide, the salt of the keratan sulfate oligosaccharide. The salt of 6-O-sulfated N-acetylheparosan and the salt of N, 6-O-sulfated heparosan.

  Here, the pharmaceutically acceptable salt is, for example, an alkali metal salt such as sodium salt, potassium salt or lithium salt, an alkaline earth metal salt such as calcium salt, a salt with an inorganic base such as ammonium salt, or diethanolamine. Examples thereof include organic base salts such as salts, cyclohexylamine salts, and amino acid salts, but are not limited thereto.

[B] Selective component The MMP inhibitor of the present invention can be blended with components other than the above-mentioned GAG oligosaccharides (1) to (5) as needed, as long as the MMP inhibitory effect is not impaired. . For example, moisturizer, cell activator, anti-inflammatory agent, antioxidant, whitening agent, hyaluronidase inhibitor, active oxygen inhibitor, antihistamine, conventional excipient, stabilizer, binder, lubricant, emulsifier, An osmotic pressure adjusting agent, a pH adjusting agent, other colorants, disintegrating agents and the like can be appropriately blended.

[C] Administration method and dosage form The MMP inhibitor of the present invention can be administered by injection (intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, etc.), oral, transdermal, inhalation, etc. Depending on the administration method, it can be appropriately formulated. The tissue or cell to which the MMP inhibitor of the present invention is administered is not particularly limited, but direct administration to the lung or bronchus is one of the preferred administration forms.

  The dosage form of the MMP inhibitor of the present invention includes, for example, injections (solutions, suspensions, emulsions, solid agents for dissolution at the time of use), tablets, capsules, granules, powders, solutions, lipolytic agents, It can be widely selected from ointments, gels, powders for external use, sprays, inhalation powders and the like.

  When the MMP inhibitor of the present invention is used as a pharmaceutical, the endotoxin concentration is preferably 0.3 EU / mL or less when used as a solution agent. The endotoxin concentration can be measured using a conventional endotoxin measurement method well known to those skilled in the art, but the Limulus test method using horseshoe crab, amebocyte, lysate components is preferred. In addition, EU (endotoxin unit) can be measured and calculated according to the Japanese Industrial Standards Biochemical Test General Rules (JIS K8008) or the US Pharmacopoeia.

  The dosage of the MMP inhibitor of the present invention depends on the purpose of administration (prevention, prevention of deterioration, improvement of symptoms, or treatment), the type of disease, the symptoms, sex, age, body weight, administration site, administration method, etc. of the patient. Although it is set and is not particularly limited, it is possible to administer 1 ng / dose site to 30 mg / dose site as the amount of GAG oligosaccharide (1) to (5) in general per adult. Is possible.

  The MMP inhibitor of the present invention can be used not only as a pharmaceutical use but also as an experimental reagent. That is, in addition to clinical trials for obtaining approval of the MMP inhibitor of the present invention as a drug, the MMP of the present invention is applied to various types of living cells in order to verify biological organs and diseases in which the MMP inhibitor of the present invention is effective. Coexisting an inhibitor to verify the presence or absence or strength of its MMP inhibitory action, or to a specific patient for an organ (for example, lung) in which the effectiveness of the MMP inhibitor of the present invention has been confirmed. It is also possible to check the presence or absence of the MMP inhibitory effect and obtain a judgment index such as the right or wrong of using the MMP inhibitor of the present invention, the dosage, and the timing of administration. In addition, in order to further fundamentally examine the microscopic or macroscopic effects of MMP inhibition on the living body, it is possible to verify the action of the MMP inhibitor of the present invention in vivo or in vitro.

  The MMP inhibitor of the present invention is particularly preferably used as an application for inhibiting MMP-9, MMP-12, and MMP-13, and more preferably used as an application for inhibiting MMP-9.

<2> Inhibition method of the present invention The inhibition method of the present invention is at least one or more selected from the group consisting of GAG oligosaccharides represented by the following formulas HA4, L4 and L4L4 and pharmaceutically acceptable salts thereof. Is a method for inhibiting MMP, which suppresses cell-derived MMP activity.

HA4: GlcA β1-3 GlcNAcβ1-4 GlcA β1-3 GlcNAc
L4; Gal6S β1-4 GlcNAc6S
L4L4; Gal6S β1-4 GlcNAc6S β1-3 Gal6S β1-4 GlcNAc6S
(In the formula, Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, GlcA represents a glucuronic acid residue, 6S represents a sulfate group at the 6-position, β1-3 represents β1- (3 glycoside bonds, β1-4 represents β1-4 glycoside bonds, respectively)

  There is no particular limitation on the species of the organism that is used in the inhibition method of the present invention and that is the source of cells that “coexist” the GAG oligosaccharides and their salts. Examples of the biological species include mammals such as humans, dogs, cats, monkeys, horses, sheep, mice, rats, rabbits and the like, but are not limited thereto. The cell type is not particularly limited as long as it is meaningful to try to inhibit the activity of MMP, and it is possible to use tumor cells in addition to various normal or non-separated normal cells. it can. In addition, the cells are preferably isolated cells.

  The means for inhibiting cell-derived MMP activity in the inhibition method of the present invention is not particularly limited. Typically, the method is performed by bringing a GAG oligosaccharide into contact with a cell to be inhibited of MMP activity. Examples of this contact include co-cultivation of cells and GAG oligosaccharide from the beginning of culture, addition of GAG oligosaccharide to cultured cells in the growth phase, addition of GAG oligosaccharide to cultured cells in the stationary phase, and the like. In addition, when it is desired to suppress the activity of MMP for a long period of time, GAG oligosaccharide can be added to the cell culture system as needed so that the concentration of GAG oligosaccharide is always kept high. Further, the conditions for contacting the cells with the GAG oligosaccharide are not particularly limited, and a temperature and pH preferable for cells in which inhibition of MMP is desired can be appropriately selected and contacted. For example, the temperature is usually in the range of about 25 to 45 ° C, preferably 30 to 40 ° C, and more preferably 35 to 38 ° C. The pH is usually in the range of about 6 to 8, and preferably 7.0 to 7.6.

  Further, the concentration of the GAG oligosaccharide when the cell is brought into contact with the GAG oligosaccharide is not particularly limited, but is usually in the range of about 0.5 mM to 20 mM, and preferably 1 mM to 5 mM. It is.

  Moreover, from the Example mentioned later, the activity of MMP can be suppressed depending on the concentration of the added GAG oligosaccharide.

  The GAG oligosaccharide and salts thereof used in the inhibition method of the present invention are the same as those of the “invention MMP inhibitor”, and the purpose and configuration of the inhibition method of the present invention are also disclosed in the “invention MMP inhibitor”. It is disclosed in a system using “cells”.

  The inhibition method of the present invention includes, for example, a patient (human or non-human) to be administered with the MMP inhibitor of the present invention, the elastase inhibitor of the present invention, or the lung disease inhibitor of the present invention containing GAG oligosaccharide as an active ingredient. In mammals) to determine if these agents are effective. That is, for example, by contacting a GAG oligosaccharide with a target patient cell isolated by biopsy or the like or a cell line established as a specific cell model, the GAG oligosaccharide It can be determined whether elastase activity can be suppressed with efficacy. If the elastase activity of the cells is effectively suppressed, the above-mentioned agent is judged to be effective against COPD in a test cell provider or a test cell line model.

<3> Elastase inhibitor of the present invention The elastase inhibitor of the present invention is an elastase activity inhibitor containing a keratan oligosaccharide as an active ingredient.

  The keratan oligosaccharide is particularly preferably a keratan sulfate oligosaccharide represented by the following formula.

L4: Gal6S β1-4 GlcNAc6S
(In the formula, Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, 6S represents a sulfate group at the 6-position, and β1-4 represents a β1-4 glycoside bond. )

  The essential component, selective component, administration method and dosage form of the elastase inhibitor of the present invention are the same as those of the “MMP inhibitor of the present invention”.

  Since elastase may be involved in immune cell migration in an inflammatory reaction, the elastase inhibitor of the present invention is also useful as an inflammation inhibitor.

<4> The therapeutic agent for lung disease of the present invention The therapeutic agent for lung disease of the present invention is a therapeutic agent for chronic obstructive pulmonary disease (COPD) containing a keratan oligosaccharide represented by the following formula as an active ingredient.

L4: Gal6S β1-4 GlcNAc6S
(In the formula, Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, 6S represents a sulfate group at the 6-position, and β1-4 represents a β1-4 glycoside bond. )

  “Treatment” in the therapeutic agent for pulmonary disease of the present invention includes a prophylactic effect capable of suppressing the occurrence of COPD in advance. In addition, these treatments are not necessarily limited to having a complete therapeutic effect, and may have a partial effect. Furthermore, the treatment target of the therapeutic agent for lung disease of the present invention is preferably emphysema.

  As a preferable route of administration of the therapeutic agent for pulmonary disease of the present invention, those exemplified as the MMP inhibitor of the present invention are preferable, and intravenous injection is particularly preferable. In addition, as described above, the essential component, selective component, administration method and dosage form of the therapeutic agent for pulmonary disease of the present invention are “the MMP inhibitor of the present invention” except that intravenous injection is particularly suitable. It is the same.

<Invention selection method>
The screening method of the present invention can be performed in substantially the same manner as the above-described inhibition method of the present invention, except that the test component is added to the cell culture system. The addition of the test component can be performed by selecting an appropriate addition concentration and appropriately combining the addition of these before cell culture, at the start of cell culture, or after cell culture. . As the cells to be used, it is preferable to use a cell line established as a specific cell model because the universality of the effect of the test drug can be confirmed more accurately.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

(Sample preparation)
(1) Preparation of L4 and L4L4 Each keratan sulfate oligosaccharide was added to a 1 mg / 200 μl aqueous solution of bovine cornea-derived keratan sulfate I (manufactured by Seikagaku Corporation) or shark cartilage-derived keratan sulfate II (manufactured by Seikagaku Corporation). Add 1 mU / μl of keratanase II diluted with 0.1 M acetate buffer, react for 2 hours at pH 5.0 and 37 ° C., and then apply a centrifugal filter (Millipore) with a molecular weight cut off of 10,000 to remove the high molecular weight component. The obtained extract is separated by GPC. The structure of the product was confirmed using a mass spectrometer (4000Q Trap, manufactured by Applied Biosystems) by the method described in JP-A-2006-292683.

(2) Preparation of HA4 HA4 was prepared by (Tawada, A, et al., Glycobiology 12, 421-426 (2002)) and (Blundell, CD, et al. Analytical Biochemistry 353, 236-247 (2006)). The preparation was carried out according to the method. Specifically, first, the chicken crown-derived HA was decomposed into oligosaccharides by sheep testis-derived hyaluronidase (manufactured by Sigma), and HA4 was prepared through purification by anion exchange chromatography and purification by GPC. The product structure was confirmed to be HA4 using a mass spectrometer as described above.

(3) Preparation of 6-O-sulfated N-acetylheparosan 6-O-sulfated N-acetylheparosan was prepared according to the method described in JP-A-2005-290383. Specifically, first, N-acetylheparosan obtained from Escherichia coli K5 strain was dissolved in distilled water, then fractionated in a column, n-tributylamine (TBA) was added, and N-acetylheparosan was added. A TBA salt was obtained. Dimethylformamide (DMF) was added to the obtained N-acetylheparosan TBA salt, and the mixture was stirred at 50 ° C. for 4 hours. Furthermore, after adding a pyridine-sulphotrioxide complex (Py-SO ₃ ), the reaction was allowed to proceed for 2 hours at 50 ° C. under stirring conditions. Thereafter, the reaction was terminated by cooling with ice, and distilled water was added and dialyzed against running water. The obtained liquid was produced, freeze-dried, dissolved in sodium acetate, fractionated on a column, and prepared by desalting and freeze-drying to obtain 6-O-sulfated N-acetylheparosan.

(4) Preparation of N, 6-O-sulfated heparosan N, 6-O-sulfated heparosan was prepared according to the method described in JP 2000-517328. Specifically, first, the polysaccharide obtained from Escherichia coli K5 strain was subjected to hydrazine decomposition and N-deacetylation, and the resulting product was N-sulfated with anhydrous / trimethylamine adduct (TMA / SO ₃ ). . Then, it melt | dissolved in distilled water and applied to the column, and TBA was added. After removing the excess of TBA with diethyl ether, was dissolved in anhydrous dimethylformamide, a Py-SO ₃ dissolved in anhydrous dimethylformamide was added, the N, 6-O-sulphated heparosan by 1 hour at 0 ℃ Obtained.

(5) Various chondroitin sulfates Various chondroitin sulfates were purchased from Seikagaku Corporation Biobusiness Corporation. Low molecular weight chondroitin sulfate was obtained by reducing the molecular weight using various chondroitin degrading enzymes. For chondroitin sulfate C, starting material chondroitin sulfate C (weight average molecular weight: 30 kDa, chondroitin sulfate C sodium salt (shark cartilage)) was used as a starting material, and 1 g was dissolved in 50 ml of PBS (pH 5.3). To this solution was added 100,000 U of sheep testicular hyaluronidase (manufactured by Sigma: type V) and an enzyme reaction was carried out at 37 ° C. A part of the reaction solution was collected over time and analyzed by GPC-HPLC to examine the degree of molecular weight reduction. When the target molecular weight was reached, the reaction solution was boiled to stop the enzyme reaction. If the target molecular weight was not reached, the same reaction was repeated until the target molecular weight was reached. The target molecular weight fraction thus obtained was added with activated carbon to the reaction-finished solution and reacted at 50 ° C. for 1 hour. After that, the reaction solution was filtered, washed with sodium acetate, ethanol was added, and the precipitate was removed. The obtained precipitate was washed with ethanol and then purified by drying. Various chondroitin sulfates with various molecular weights were obtained as white powders by the above method.

[Example 1] Establishment of activity measurement system An MMP activity measurement system using a serum-free culture supernatant of macrophage cells and a specific substrate was established. Macrophage cells are known to be the main MMP producing cells in vivo. The culture supernatant was concentrated and a total protein amount of 14-18 mg / ml was used as the MMP enzyme solution. The culture supernatant contained about 0.01% and 0.0006% of MMP-9 and MMP-13, respectively. This time, with the aim of screening for the inhibitory effect on total MMP activity with the aim of developing therapeutic agents, a general-purpose MMP substrate [DNP-Pro-Cha-Gly-Cys (Me) -His-Ala-Lys (N- Me-Abz) -NH ₂ (Cha = β-cyclohexylalanyl, Abz = 2-aminobenzoyl (anthraniloyl) (Ex. 365 nm / Em. 450 nm))] (SEQ ID NO: 1) was adopted. The substrate has been degraded by MMP-1, 3, 7, 8, 9, 11, 12, 13, 14 and is reported to have kcat / Km = 1.3 × 10 ⁴ for MMP-9. When examined using MMP-9 purified enzyme, the measurement sensitivity using this substrate was 0.015 ng or more.

[Example 2] Changes in MMP activity by GAG oligosaccharide First, 10 µl of each of the various GAG sugar chains (final concentration 1 mg / ml) prepared above and GM6001 (Cosmo Bio Co., Ltd.), a commercially available MMP inhibitor as a positive control. Purchased from the company: final concentration 25 μM), human-derived recombinant MMP-9 (4 ng / ml) 10 μl, purified water 20 μl and buffer (final concentration 50 mM Tris-HCL, pH 7.6, 1.5 mM NaCl, 0.5 mM CaCl ₂ , 1 [mu] M ZnCl _2, were mixed 0.01% (v / v) Brij -35) 50μl, was incubated for 1 hour at 37 ° C.. In addition, the negative control used what did not mix | blend the GAG sugar chain which should be mix | blended with each type | system | group.

Then, after adding 10 μl of fluorescently labeled MMP substrate, the fluorescence intensity was measured up to 120 minutes at 2-minute intervals in a 96-well plate, and the decomposition product concentration was determined. The substrate is DNP-Pro-Cha-Gly-Cys (Me) -His-Ala-Lys (N-Me-Abz) -NH ₂ (Cha = β-cyclohexylalanyl, Abz = 2-aminobenzoyl (anthraniloyl) (Ex 365 nm / Em. 450 nm)) (SEQ ID NO: 1) was used at a final concentration of 4 μM.

  MMP inhibitory activity was calculated as a specific activity value (%), assuming that the activity without inhibitor was 100%. The influence on MMP activity according to various GAG oligosaccharide groups was examined. From these results, a low-molecular-weight HA oligosaccharide, keratan sulfate oligosaccharide, sulfated heparosan, and 6-O-sulfated-N-acetylheparosan were significantly inhibited by MMP activity (FIG. 1). In FIG. 1, the vertical axis represents specific activity values, and each bar on the horizontal axis represents these activities in each GAG oligosaccharide. Along with the results shown in FIG. 1, FIG. 2 shows the results of examining the degree of inhibition of MMP-9 by changing the number of linked oligosaccharides of HA oligosaccharide. In FIG. 2, “Medium” is a negative control and “GM6001” is a positive control. “4” indicates “HA4”, “6” indicates “HA6”, “8” indicates “HA8”, “10” indicates “HA10”, and “12” indicates “HA12”. In addition, “5 kDa”, “25 kDa”, and “200 kDa” indicate that the molecular weight is hyaluronic acid, respectively. From the results of FIG. 2, among hyaluronic acid oligosaccharides, MMP inhibitory activity was observed in HA4 or HA6, and it became clear that HA4 is most suitable.

  Furthermore, when the relationship between the concentration dependency of chondroitin sulfate C oligosaccharide (CS-C) having excellent MMP inhibitory activity and the degree of sugar polymerization was examined using MMP-9 purified enzyme, L4 and L4L4 were particularly suppressed. It was found that the effect was high and the MMP activity was inhibited in a concentration-dependent manner (FIG. 3). FIG. 3 (1) shows the concentration-dependent MMP inhibitory activity in L4, and FIG. 3 (2) shows the concentration-dependent MMP inhibitory activity in L4L4. The vertical axis in FIG. 3 is the specific activity value (%), and the horizontal axis is the concentration of each GAG oligosaccharide added. When L4L4 was measured for inhibition constant Ki for MMP-9, it was 0.8 mM for L4 and 0.6 mM for L4L4, respectively. When the commercially available MMP inhibitor GM6001 used as a positive control was measured by the same method, its Ki was about 4 μM (FIGS. 4A to 4C). FIG. 4A is a drawing showing the Ki for the commercially available MMP inhibitor GM6001, FIG. 4B is a data analysis diagram based on the calculation of Ki values for L4, and FIG. 4C for L4L4. In each case, the vertical axis represents the gradient, and the horizontal axis represents the amount of GAG oligosaccharide added.

Example 3 Elastase Induction Model A 5 mg / ml aqueous solution was intravenously injected into 9 to 11 week-old C57BL / 6 mice (male: purchased from Nippon Claire Co., Ltd.) so that L4 was 250 μg / body. As a control, a group administered with 50 μl of physiological saline was used. 30 minutes later, elastase 3U derived from porcine pancreas was sprayed into the trachea. 24 hours after administration of elastase, the lungs were dissected and bronchoalveolar lavage was performed 3 times with physiological saline on the lungs to obtain bronchopulmonary lavage fluid (BALF). Evaluation items were evaluated based on the total cell count and neutrophil content in bronchopulmonary lavage fluid. The results are shown in FIGS. FIG. 5A is a drawing examining the effect of L4 administration on the total cell number, and FIG. 5B is a drawing examining the effect of L4 administration on the neutrophil count. 5A and 5B, the vertical axis represents the number of cells, and the bar graph on the vertical axis represents the test system. FIG. 5C is a diagram showing the ratio of neutrophils in the calculated total number of cells (%: vertical axis). As shown in these figures, it was found that the neutrophil accumulation was significantly suppressed in the group administered with L4 prior to the administration of elastase.

  In this experiment, activity inhibition effect of MMP-9 and the like was recognized, and the enhancement of the migration ability of neutrophils, which are MMP-producing cells, was suppressed in elastase-induced pulmonary emphysema model mice, so L4 should be used as a COPD therapeutic agent. It is thought that you can.

[Example 4] Elastase-induced pulmonary emphysema model 9 to 11-week-old C57BL / 6 mice (male: purchased from CLEA Japan, Inc.) were intravenously injected with a 5 mg / ml aqueous solution so that L4 was 250 µg / body. . As a control, a group administered with 50 μl of physiological saline was used (first L4 administration). Thirty minutes after the first L4 administration, 5 U of porcine pancreatic elastase was sprayed into the trachea. 24 hours after this elastase administration, L4 administration having the same contents as the first administration was carried out together with 50 μl of physiological saline in the control group (second administration of L4). 24 hours after the second L4 administration, a 5 mg / ml aqueous solution was intravenously administered so that L4 was 125 μg / body, and 50 μl of physiological saline was administered in the control group (third L4 administration). . Twenty-one days after the third L4 administration, the mice subjected to the experiment were sacrificed, formalin fixation of the lungs was performed according to a conventional method, and the average distance between the alveolar walls (Lm) of the lungs was measured. The number of mice in the L4 administration group (L4) is 9, the number of mice in the saline administration group (PS) is 9, and the number of n in the untreated group (Untreat) is 2. The test was conducted.

  The results of this emphysema model test are shown in FIG. 6 (A, B). The vertical axis of the bar graph in FIG. 6A indicates the average alveolar wall distance (Lm) of the lungs, and the numbers straddling between the bars are p-values indicating the probabilistic risk factors. The results presented here revealed that L4 significantly suppresses elastase-induced emphysema. FIG. 6B discloses formalin fixation images of the lungs of the mice of each test group, but the lung tissue of the L4 group is denser than the PS group, and the numerical results shown in FIG. 6A It is what supports.

Claims (6)

MMP- containing, as an active ingredient, at least one or more selected from the group consisting of glycosaminoglycan oligosaccharides represented by the following formulas HA4, L4 and L4L4 and pharmaceutically acceptable salts thereof 9 inhibitor .
HA4: GlcA β1-3 GlcNAc β1-4 GlcA β1-3 GlcNAc
L4: Gal6S β1-4 GlcNAc6S
L4L4: Gal6S β1-4 GlcNAc6S β1-3 Gal6S β1-4 GlcNAc6S
(In the formula, Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, GlcA represents a glucuronic acid residue, 6S represents a sulfate group at the 6-position, β1-3 represents β1- 3 represents a glycosidic bond, and β1-4 represents a β1-4 glycosidic bond. Formula HA4, L4, by at least one or more selected from the group consisting of salts glycosaminoglycan oligosaccharides and pharmaceutically acceptable represented respectively L4L4, isolated from cells MMP- 9 Method for inhibiting MMP-9 , which suppresses activity .
HA4: GlcA β1-3 GlcNAc β1-4 GlcA β1-3 GlcNAc
L4: Gal6S β1-4 GlcNAc6S
L4L4: Gal6S β1-4 GlcNAc6S β1-3 Gal6S β1-4 GlcNAc6S
(In the formula, Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, GlcA represents a glucuronic acid residue, 6S represents a sulfate group at the 6-position, β1-3 represents β1- (3 glycoside bonds, β1-4 represents β1-4 glycoside bonds, respectively) An elastase-induced emphysema inhibitor comprising a keratan sulfate oligosaccharide represented by the following formula L4 as an active ingredient.
L4: Gal6S β1-4 GlcNAc6S
(In the formula, Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, 6S represents a sulfate group at the 6-position, and β1-4 represents a β1-4 glycoside bond. ) A therapeutic agent for chronic obstructive pulmonary disease comprising a keratan sulfate oligosaccharide represented by the following formula L4 as an active ingredient.
L4: Gal6S β1-4 GlcNAc6S
(In the formula, Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, 6S represents a sulfate group at the 6-position, and β1-4 represents a β1-4 glycoside bond. ) The therapeutic agent according to claim 4 , wherein the chronic obstructive pulmonary disease is emphysema. Contacting a cell with at least one or more selected from the group consisting of glycosaminoglycan oligosaccharides represented by the following formulas HA4, L4 and L4L4 and pharmaceutically acceptable salts thereof, and a test drug And selecting a drug that promotes or alleviates suppression of MMP-9 activity derived from the cell by the glycosaminoglycan oligosaccharide or salt .
HA4: GlcA β1-3 GlcNAc β1-4 GlcA β1-3 GlcNAc
L4: Gal6S β1-4 GlcNAc6S
L4L4: Gal6S β1-4 GlcNAc6S β1-3 Gal6S β1-4 GlcNAc6S
(In the formula, Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, GlcA represents a glucuronic acid residue, 6S represents a sulfate group at the 6-position, β1-3 represents β1- (3 glycoside bonds, β1-4 represents β1-4 glycoside bonds, respectively)

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