JP2006151843A - Cathepsin k inhibitor and food imparted with the function of the inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cathepsin K inhibitor having excellent safety and applicable to foods as well as medicines, and provide a composition or food usable for the treatment, prevention, etc., of diseases or conditions induced by the increase of bone loss or bone resorption. <P>SOLUTION: The invention relates to a cathepsin K inhibitor containing a protein of a plant of the family Gramineae as an active component and to a composition or food containing a protein of a plant of the family Gramineae as an active component and usable for the treatment, prevention or amelioration of diseases or conditions which can be treated, prevented or ameliorated by the inhibition of cathepsin K. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

Background of the Invention

FIELD OF THE INVENTION The present invention relates to an inhibitor having cathepsin K inhibitory activity and a food provided with the function. More specifically, the inhibitor has a component derived from a natural product as an active ingredient. The present invention also relates to a composition for use in the treatment, prevention or amelioration of a disease or condition that can be treated, prevented or ameliorated by inhibiting cathepsin K.

BACKGROUND ART With the progress of an aging society, the increase of “bedridden elderly people” has become a major social and economic problem. The main causes of “old bedridden people” are strokes, old age, and fractures caused by osteoporosis. In particular, prevention of osteoporosis is an important issue in maintaining and improving the quality of life (QOL) of the elderly.

  It is known that bone mass in the body is maintained by a balance between bone formation by mesenchymal osteoblasts and bone resorption by hematopoietic osteoclasts. If this balance breaks down and the level of bone resorption exceeds bone formation, bone mass may decrease and ultimately osteoporosis. In addition, it is known that increased bone resorption is associated with not only osteoporosis but also diseases such as osteoarthritis and rheumatoid arthritis, and is an important target for preventing or treating these diseases. It is the osteoclast.

  Osteoclasts are multinucleated giant cells that attach to the bone surface and form resorption pits between the wavy edge and the bone surface. In this resorption fossa, osteoclasts decompose proteins such as type I collagen with protease and dissolve hydroxyapatite with the acid to be released. It has been reported that proteases expressed by osteoclasts are diverse, such as cathepsin B, cathepsin H and cathepsin L. Among them, cathepsin K is attracting attention in relation to various pathological conditions.

  Cathepsin K is a kind of cysteine protease, and its mRNA is selectively expressed at high levels in osteoclasts [J. Biol. Chem. (1996) 271: 12511] (Non-patent document 1), cathepsin. It has been reported that K is localized specifically in osteoclasts [Bone (1997) 20:81] (Non-patent Document 2). It has also been reported that an antisense oligonucleotide of cathepsin K inhibits bone resorption by osteoclasts [J. Biol. Chem. (1997) 272: 8109; Mol. Carcinog. (2001) 32: 84] (Non-Patent Documents 3 and 4 respectively), the importance of cathepsin K in bone resorption has been pointed out. In addition, cathepsin K is an osteoclastoma [Biol. Chem. Hoppe-Seyler (1995) 376: 379], bone erosion in cholesteatoma [Mod. Pathol. (2001) 14: 1226], prostate cancer metastasis [ J. Bone Miner. Res. (2003) 18: 222], obesity [J. Cell Physiol. (2003) 195: 309], pulmonary fibrosis [Am. J. Pathol. (2004) 164: 2203], and It is speculated that it plays an important role in pathophysiology such as giant cell tumor of bone [Am. J. Pathol. (2004) 165: 593].

  For this reason, cathepsin K has been attracting attention as a target molecule for disease treatment and prevention, and research and development of cathepsin K inhibitors have been actively conducted. So far, as cathepsin K inhibitors, for example, dipeptidyl ketone inhibitors [Bioorg. Med. Chem. (1999) 7: 581], diaminopyridinone inhibitors [Bioorg. Med. Chem. Lett. (1999) 9 : 1907], diacylcarbohydrazide inhibitors [Biochemistry (1999) 38: 15893], pyridoxal propionate derivatives [Biochem. Biophys. Res. Commun. (2000) 267: 850], cyclic ketone derivatives [J. Med] Chem. (2001) 44: 725], allylaminoethylamide derivatives [J. Med. Chem. (2002) 45: 2352; Bioorg Med. Chem. Lett. (2003) 13: 1997], 4-amino-azepane. -3-one inhibitors [Special Table 2003-533432], 3-allylamino-azetidin-2-one inhibitors [Bioorg. Med. Chem. Lett. (2003) 13: 139] and ketoamide derivatives [Bioorg. Med. Chem] Lett. (2004) 14: 4897] has been reported.

However, all of these known cathepsin K inhibitors are synthetic compounds, and there was anxiety from the viewpoint of safety when taking them in forms other than pharmaceuticals. For this reason, it can be said that it was difficult to apply it to daily intake through foods.
J. Biol. Chem. (1996) 271: 12511 Bone (1997) 20:81 J. Biol. Chem. (1997) 272: 8109 Mol. Carcinog. (2001) 32:84

Summary of the Invention

The present inventors have now found that a protein contained in a gramineous plant such as rice ( Oryza sativa ) has an excellent activity of inhibiting cathepsin K. It has never been known at all that a protein contained in a grass family has an activity to inhibit cathepsin K. It can be said that it was unexpected that the protein contained in the gramineous plant has the activity to inhibit cathepsin K. The present inventors have also found that these proteins having the activity of inhibiting cathepsin K are mainly oryzasistatins such as oryzastatin-I and oryzasistatin-II. Furthermore, the present inventors also discovered oryzasistatin-III as a novel protein having an activity of inhibiting cathepsin K. The present invention is based on these findings.

  Therefore, the present invention provides a cathepsin K inhibitor that is excellent not only as a pharmaceutical product but also excellent in safety and can be applied to foods, and treatment or prevention of a disease or condition that can be induced by bone loss or enhanced bone resorption. The purpose of the present invention is to provide a composition or food used in the above.

Cathepsin K inhibition according to the present invention comprises a protein contained in a grass family plant as an active ingredient.
According to the present invention, a composition comprising, as an active ingredient, a protein contained in the aforementioned gramineous plant, which is used for treatment, prevention, or improvement of a disease or condition that can be treated, prevented, or ameliorated by inhibiting cathepsin K. Is provided.

According to another aspect of the present invention, the food according to the present invention is a food comprising an effective amount of the above protein, and is capable of treating, preventing or ameliorating a disease or condition that can be treated, prevented or ameliorated by inhibiting cathepsin K. It is a food used in
According to still another aspect of the present invention, the food according to the present invention is a food comprising an effective amount of the protein and used for cathepsin K inhibition.
According to another aspect of the present invention, there is provided oryzasistatin-III protein and homologues thereof.

  The active ingredient used in the present invention is derived from a gramineous plant, and the gramineous plant is often ingested as a staple food cereal, for example, like rice, and has abundant experience as a food. Therefore, it can be said that there is almost no problem in terms of safety. For this reason, the cathepsin K inhibitor and composition by this invention can be used for uses, such as not only a pharmaceutical but foodstuffs including a quasi-drug and a functional food. Therefore, the cathepsin K inhibitor, the composition and the food according to the present invention have few side effects, are relatively inexpensive and highly safe even if they are taken or ingested over a long period of time.

Detailed description of the invention

Active ingredient and production method thereof As described above, the cathepsin K inhibitor according to the present invention and the active ingredient of food are proteins contained in the grass family. Here, the gramineous plant refers to a plant belonging to the gramineous family in plant taxonomy, and examples thereof include rice, wheat, barley, corn, millet, and millet. Preferably, the gramineous plant is rice ( Oryza sativa ).

  According to a preferred embodiment of the present invention, the protein contained in the grass plant which is an active ingredient is oryzasistatin-I, oryzasistatin-II, oryzatistatin-III, or a partial peptide thereof.

Here, oryzasistatin-I has the following amino acid sequence:
MSSDGGPVLGGVEPVGNENDLHLVDLARFAVTEHNKKANSLLEFEKLVSVKQQVVAGTLYYFTIEVKEGDAKKLYEAKVWEKPWMDFKELQEFKPVDASANA (SEQ ID NO: 1)
It is reported that it is a rice protein having a molecular weight of 11,000 as defined in the above and is a cysteine protease inhibitor that inhibits the activity of proteases such as papain [Agric. Biol. Chem. (1987) 51: 2763, J. Biol. Chem. (1987) 262: 16793].

Orizacystatin-II has the following amino acid sequence:
MAEEAQSHAREGGRHPRQPAGRENDLTTVELARFAVAEHNSKANAMLELERVVKVRQQVVGGFMHYLTVEVKEPGGANKLYEAKVWERAWENFKQLQDFKPLDDATA (SEQ ID NO: 2)
It is reported that it is a cysteine protease inhibitor that inhibits the activity of proteases such as papain in the same manner as oryzasistatin-I [J. Biol. Chem. 1990) 265: 15832].

  Regarding the cysteine protease inhibitory activity of oryzasistatin-I and II, it has been reported that papain, ficin and cathepsin H are inhibited, whereas cathepsin B and cathepsin L belonging to cysteine protease are not inhibited [J. Biol. Chem. 265: 15832-15837, 1990]. Similarly, it has been reported that sugarcane cystatin inhibits papain, cathepsin L, cathepsin K and the like, but not ficin and bromelain belonging to cysteine protease [Biochem. Biophys. Res. Commun. 320: 1082-1086, 2004]. Therefore, since it has other cysteine protease inhibitory activities such as papain, it is difficult to predict the presence and level of cathepsin K inhibitors, and oryzasistatin I and II have cathepsin K inhibition. Was unexpected.

Furthermore, oryzasistatin-III has the amino acid sequence:
MAEEAQQPRGVKVGGIHDAPAGRENDLTTVELARFAVAEHNSKANAMLELERVVKVRQQVVGGFMHYLTVEVKEPGGANKLYEAKVWERAWENFKQLQDFKPLDDATA (SEQ ID NO: 3)
A rice protein having a molecular weight of 12,000 as defined in (1), wherein the protein was obtained for the first time and the sequence was identified.

  Here, the “partial peptide” is a peptide consisting of a part of the entire amino acid sequence of the protein that is the active ingredient of the present invention, and at least includes an active site region capable of exhibiting cathepsin K inhibitory activity of the protein. Say things. "Partial peptide" is obtained in the process of producing a protein as an active ingredient, or the obtained protein is cleaved by a conventional amino acid sequence cleaving means, and the presence or absence of activity is confirmed by an activity test described later, Obtainable.

  In the present specification, the term protein refers to a protein derivative. Here, the protein derivative has the cathepsin K inhibitory activity described above, and is part of the amino group at the amino terminus (N terminus) of the amino acid sequence of the protein or the amino group at the side chain of each amino acid. Alternatively, all and / or the carboxyl group at the carboxyl terminus (C-terminus) of the amino acid sequence of the protein or part or all of the carboxyl groups of the side chains of various amino acids, and / or the amino acids of the side chains of the respective amino acids of the protein A functional group (for example, a hydrogen group, a thiol group, an amide group, etc.) other than a group and a carboxyl group is partially or entirely modified with another appropriate substituent (for example, a phosphate group). Such modifications with other appropriate substituents may be performed for the purpose of, for example, protecting functional groups present in proteins, improving safety and tissue migration, or enhancing activity.

  Alternatively, the protein derivatives include pharmaceutically acceptable salts of the protein according to the present invention. Preferable examples of such salts include alkali metal or alkaline earth metal salts such as sodium salt or calcium salt, hydrohalides such as hydrofluoride and hydrochloride, nitrates, sulfates, phosphorus Inorganic acid salts such as acid salts, lower alkyl sulfonates such as ethane sulfonate, aryl sulfonates such as benzene sulfonate, organic acid salts such as fumaric acid, succinate, acetate, and Examples include amino acid salts. Furthermore, the protein according to the present invention may be a solvate. Such solvates include hydrates, alcohol solvates (eg, methanol solvates, ethanol solvates), and ether solvates.

  In this specification, amino acids include both optical isomers, that is, D-form and L-form. The amino acids here include not only 20 kinds of α-amino acids constituting natural proteins, but also other α-amino acids, β-, γ-, δ-amino acids and unnatural amino acids. May be.

According to one preferred embodiment of the present invention, the protein as the active ingredient can be selected from the group consisting of the following (a) to (d):
(A) a protein comprising an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 3,
(B) a partial peptide consisting of a part of the amino acid sequence of (a), having a cathepsin K inhibitory activity,
(C) a protein comprising an amino acid sequence having at least 90% homology with the protein comprising the amino acid sequence of (a) and having cathepsin K inhibitory activity, and (d) the above (a) A protein comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted, added or inserted, and having cathepsin K inhibitory activity.

Furthermore, according to another aspect of the present invention, there is provided a protein selected from the group consisting of the following (a ′) to (d ′).
(A ′) a protein comprising an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 3;
(B ′) a partial peptide consisting of a part of the amino acid sequence of (a ′), which has a cathepsin K inhibitory activity,
(C ′) a protein comprising an amino acid sequence having at least 90% homology with the protein comprising the amino acid sequence of (a ′) and having cathepsin K inhibitory activity, and (d ′) A protein comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted, added or inserted in the amino acid sequence of (a ′) and having cathepsin K inhibitory activity.

The protein of (c) or (c ′) consists of an amino acid sequence having at least 90% homology with the protein of the amino acid sequence of (a) or (a ′), wherein the homology is Preferably, it is 95% or more, more preferably 98% or more.
In addition, any numerical value of homology shown in the present specification may be any numerical value calculated using a homology search program known to those skilled in the art. For example, in FASTA, BLAST (Basic Local Alignment Search Tool), etc. By using default (initial setting) parameters, it can be easily calculated.

  Here, “cathepsin K inhibitory activity” refers to a property capable of inhibiting the activity of cathepsin K in vivo. Having “cathepsin K inhibitory activity” is evaluated, for example, that cathepsin K inhibitory activity was observed when measured under the same conditions as in the section of the evaluation test “measurement of cathepsin K inhibitory activity” in Examples described later. Means the case.

  In the above (d) or (d ′), the number of amino acid residues that may be deleted, substituted, inserted or added to the amino acid sequence shown in SEQ ID NOs: 1 to 3 is preferably 1 to 5. More preferably, it is 1 to 3, more preferably 1 to 2, and still more preferably 1.

  According to a preferred embodiment of the present invention, the protein of (d) or (d ′) has one or several amino acid residues conservatively substituted in the amino acid sequence of (a) or (a ′). And having cathepsin K inhibitory activity.

  As used herein, “conservative substitution” means that one or more amino acid residues are replaced with another chemically similar amino acid residue so as not to substantially alter the activity of the peptide. For example, when a certain hydrophobic residue is substituted by another hydrophobic residue, a certain polar residue is substituted by another polar residue having the same charge, and the like. Functionally similar amino acids that can make such conservative substitutions are known in the art for each amino acid. Specifically, examples of nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, and the like. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine and the like. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid. The peptides according to the present invention are usually composed of polar (neutral) amino acids.

  In the present invention, the protein as an active ingredient can be obtained by separating a protein fraction from extracts from leaves, stems, skins, roots, seeds, and the like of gramineous plants according to a conventional method. Here, the method for producing the extract is not particularly limited. For example, the extract can be used to remove plant parts such as leaves, stems, skins, roots, and seeds of grasses such as water or phosphate buffer. After triturating in a solvent, the supernatant can be collected by filtration or centrifugation, and this can be obtained by diluting with a solvent if necessary. The method for separating and purifying the protein fraction from the extract is not particularly limited. For example, ammonium sulfate is added to the extract, the precipitated protein is collected by centrifugation, and a phosphate buffer is used. It can be separated and purified by removing it as a low molecular fraction by applying dialysis or gel filtration. As described above, the active ingredient may be purified from the desired protein as described above, but if it contains an effective amount of the active ingredient protein, the plant tissue or its dried product, pulverized product, extraction You may use a thing as an active ingredient as it is. In addition, the preparation method of a dried product or a pulverized product is not particularly limited as long as the active ingredient according to the present invention can be obtained.

  Furthermore, when the amino acid sequence of the protein, which is an active ingredient in the present invention, is known in advance, it can be synthesized according to a conventional peptide synthesis technique. For example, the amino acid sequences of oryzasistatin I to III are as described above. Therefore, the protein can be obtained by chemical synthesis.

  The protein according to the present invention may be produced by a genetic engineering technique. That is, the protein according to the present invention can be produced in a transformed cell obtained by transforming a host cell with the DNA, when DNA encoding the amino acid sequence indicating it can be obtained or produced. . That is, the protein according to the present invention is in the form of a DNA, particularly a recombinant vector, which contains a DNA fragment encoding the amino acid sequence indicating it in a state in which it can be replicated in a host cell and the gene can be expressed. It can be produced by transforming a host cell and culturing the resulting transformant. At this time, a so-called host-vector system may be used for the production of the protein. In applying such a host-vector system, various expression vector (recombinant vector) production methods and transformation methods commonly used in this technical field can be used.

Use The protein which is an active ingredient by this invention has the activity which inhibits cathepsin K (section of the evaluation test "measurement of cathepsin K inhibitory activity" of an Example).

  Cathepsin K, as described above, (i) that its mRNA is selectively expressed at high levels in osteoclasts [J. Biol. Chem. (1996) 271: 12511], and (ii) cathepsin K Has been reported to be specifically localized to osteoclasts [Bone (1997) 20:81]. In addition, it has been reported that inhibition of cathepsin K inhibits bone resorption by osteoclasts [J. Biol. Chem. (1997) 272: 8109; Mol. Carcinog. (2001) 32:84]. This suggests that inhibiting cathepsin K is effective in preventing or treating diseases related to bone resorption. Regarding cathepsin K inhibitors, as a result of examining the action of SB331750, which is a cathepsin K inhibitor, in rats, it has been reported that SB331750 inhibits bone resorption [Bone (2002) 30: 746-53]. This document also suggests that it is effective to inhibit cathepsin K for the prevention or treatment of diseases related to bone resorption. Therefore, when cathepsin K is inhibited, bone loss or bone resorption enhancement is inhibited or suppressed, and diseases that can be induced by such enhancement, such as osteoporosis, osteoarthritis, rheumatoid arthritis, etc. It is possible to treat, prevent, ameliorate, alleviate the condition, or delay its progression. Furthermore, cathepsin K may play an important role in pathophysiology such as osteoclastoma, bone erosion in cholesteroma, metastasis of prostate cancer, obesity, pulmonary fibrosis, and giant cell tumor of bone. As reported in the literature shown in the above-mentioned prior art section, it has been suggested that inhibition of cathepsin K is effective in the treatment, prevention, improvement and alleviation of the condition of tumor metastasis and obesity. . Therefore, since the active ingredient according to the present invention has an activity to inhibit cathepsin K (Evaluation Test of Example, “Measurement of Cathepsin K Inhibitory Activity”), it can be used for the purpose of inhibiting cathepsin K. The active ingredient according to the present invention can be used for the treatment, prevention or amelioration of a disease or condition that can be treated, prevented or ameliorated by inhibiting cathepsin K.

  In the present specification, “treatment, prevention or amelioration” of a disease or condition is used in a sense including regulation, delay of progression, alleviation, prevention of onset, prevention of recurrence, suppression of the disease or condition.

  Here, diseases or conditions that can be treated, prevented, or ameliorated by inhibition of cathepsin K include diseases or conditions that can be induced by bone loss or enhanced bone resorption, tumor metastasis, obesity, and the like. More specifically, diseases or conditions that can be induced by bone loss or increased bone resorption include, for example, osteoporosis, osteoarthritis, rheumatoid arthritis, or conditions related thereto. Preferably, the disease or condition is osteoporosis, osteoarthritis, rheumatoid arthritis, or a condition related thereto.

  According to another aspect of the present invention, there is provided a method for inhibiting cathepsin K, comprising administering to a mammal an effective amount of a protein that is an active ingredient of the present invention. Here, the “effective amount” is an amount sufficient to exert cathepsin K inhibitory activity in a desired region in the body by administration.

  According to another embodiment of the present invention, it can be treated, prevented or ameliorated by inhibiting cathepsin K, comprising administering to a mammal a therapeutically effective amount of a protein which is an active ingredient of the present invention. Methods of treating, preventing or ameliorating treatment of a disease or condition are provided. As used herein, “therapeutically effective amount” is an amount sufficient to ameliorate one or more symptoms usually associated with the disease or condition for which treatment is desired. When referring to prophylactic use, the term means an amount sufficient to prevent or delay the onset of the disease or condition.

Composition and Food As described above, the composition according to the present invention comprises a protein contained in the aforementioned gramineous plant as an active ingredient.
Here, “comprising as an active ingredient” may contain a physiologically acceptable carrier according to a desired product form, and may contain other auxiliary ingredients that can be used in combination. It means to include. That is, the composition according to the present invention can be produced by mixing a protein as an active ingredient with a physiologically acceptable carrier, excipient, binder, diluent and the like. The composition according to the present invention can be administered or ingested orally or parenterally. Examples of oral forms include foods, granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. Examples of parenteral forms include injections (eg, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections), drops, and external preparations (eg, nasal preparations, transdermal preparations, Ointment) and suppositories (for example, rectal suppositories, vaginal suppositories). These preparations can be formulated together with a pharmaceutically acceptable carrier (for example, an excipient or an additive) by a technique usually performed in the art. Examples of pharmaceutically acceptable excipients and additives include carriers, binders, fragrances, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, and the like. It is done. Examples of the pharmaceutically acceptable carrier include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter and the like. It is done.

The preparation can be produced, for example, as follows. An oral preparation is given as an example of formulation.
Oral preparations contain active ingredients such as excipients (eg lactose, sucrose, starch, mannitol), disintegrants (eg calcium carbonate, carboxymethylcellulose calcium), binders (eg pregelatinized starch, gum arabic, carboxy Methylcellulose) or lubricants (eg, talc, magnesium stearate, polyethylene glycol) and compression molded, and if necessary, known methods for taste masking, enteric or persistent purposes It can be manufactured by applying and coating. As the coating agent, for example, known ones such as ethyl cellulose and hydroxymethyl cellulose can be used.

In formulating, one or more medically effective active ingredients other than the active ingredient according to the present invention may be further added and blended. In administration of the active ingredient according to the present invention, one or more kinds of medically effective active ingredients other than the active ingredient according to the present invention may be administered in combination.
Moreover, the form of a formulation can be suitably selected according to the target disease or condition. The administration route can also be appropriately selected according to the form of the preparation, the type of disease, the purpose of treatment, and the like.

  The compositions and inhibitors according to the invention are intended not only for pharmaceutical applications but also for foods. In the application of the composition and the inhibitor of the present invention to foods, reference can be made to the description relating to foods described below.

The food according to the present invention comprises an effective amount of the active ingredient according to the present invention.
Here, “comprising an effective amount of an active ingredient” means that the active ingredient is contained in such an amount that the active ingredient is ingested in the range described later, as a result of ingesting the amount of each food or drink normally consumed. To do. In the food according to the present invention, the active ingredient according to the present invention can be blended in the food as it is or in the form of the composition or inhibitor as described above. More specifically, the food according to the present invention is prepared by directly mixing the active ingredient according to the present invention as a food, various proteins, sugars, fats, trace elements, vitamins and the like, It may be a liquid, semi-liquid or solid, a paste, or a general food or drink.

  In the present invention, the “food” is not a pharmaceutical and is not particularly limited as long as it can be ingested by mammals. The form of the food is either liquid, semi-liquid or solid. Also good. “Food” includes foods classified as health foods, functional foods, foods for specified health use, dietary supplements, foods with a disease risk reduction label, or foods for the sick.

  The active ingredient according to the present invention has a disease or condition that can be induced by bone loss or increased bone resorption, tumor metastasis, obesity prevention, amelioration, or suppression of progression. For this reason, the active ingredient of the present invention is added to foods, health foods, functional foods and supplements (for example, foods containing one or more vitamins such as minerals such as calcium and magnesium and vitamin K) taken in daily life. In combination, a food or a disease or condition that can be induced by bone loss or increased bone resorption, tumor metastasis, obesity prevention and improvement functions can be provided.

  As described above, according to the present invention, there is provided a food comprising an effective amount of the protein, which is used for the treatment, prevention or improvement of a disease or condition that can be treated, prevented or ameliorated by inhibiting cathepsin K. Provided. According to another aspect of the present invention, as described above, a food comprising an effective amount of the protein and used for cathepsin K inhibition is provided.

According to another aspect of the present invention, a food comprising an effective amount of the protein, and a function of treating, preventing or ameliorating a disease or condition that can be treated, prevented or ameliorated by inhibiting cathepsin K is displayed. Food is provided. According to still another aspect of the present invention, there is provided a food comprising an effective amount of the protein and displaying a cathepsin K inhibitory ability.
Here, the function indications attached to these foods can be any of the main body of the product, the container, the packaging, the instructions, the package insert, or the promotional material.

  Thus, the food according to the present invention is suitable for, for example, a food suitable for a consumer who expects an improvement or alleviation of a condition associated with bone loss or increased bone resorption, or a consumer who expects an improvement or alleviation of obesity. Food, that is, so-called food for specified health use. The food for specified health here refers to a disease or condition that can be induced by bone loss or increased bone resorption, tumor metastasis, prevention, improvement of obesity, relief of condition, etc. This means food that may be subject to some legal restrictions in each country for health reasons. Such foods can also be foods that indicate that the foods may reduce disease risk, i.e., foods with a disease risk reduction label. Here, the disease risk reduction label is a label for foods that may reduce the disease risk, and is based on or based on the standard established by the FAO / WHO Joint Food Standards Committee (Codex Committee). The display can be a defined or recognized display with reference to FIG.

  Specific examples of foods according to the present invention include foods containing carbohydrates such as rice, noodles, breads and pasta; Western confectionery such as cookies and cakes; rice confectionery; Japanese confectionery such as buns and mutton; candy and gums , Various confectioneries such as frozen confectionery such as yogurt and pudding; ice confectionery; various beverages such as juice, soft drink, milk beverage, tea beverage, functional beverage, nutritional supplement beverage, non-alcohol beer; alcohol such as beer and sparkling liquor Beverages; seasonings; processed products using eggs, seafood, livestock meat, and the like.

  Preferred examples of foods to which the active ingredient of the present invention is added / blended include beverages and powdered beverages.

  Examples of the forms of health foods and functional foods to be ingested as supplements that are targets for addition and blending of the active ingredients of the present invention include beverages such as juice and tea, jelly, capsules, granules, granules, and pastes. Can be mentioned. Cathepsin K inhibitory action by processing the active ingredient of the present invention alone or in combination with other ingredients (for example, plant material) into a form of beverage, jelly, capsule, granule, granule, paste, etc. Alternatively, it can be provided as a health food or a functional food such as a supplement having a function of improving or alleviating a condition related to these actions. In particular, it is possible to prevent, ameliorate, or alleviate a state that is prevented or ameliorated by cathepsin K inhibition by combining with other components other than the active ingredient according to the present invention that are considered to have cathepsin K inhibitory activity. Functions can be further enhanced. Such other components can be selected from known components such as the compounds shown in the prior art section.

  The protein that is an active ingredient of the present invention is a component derived from the grass family that humans have taken as a staple food cereal for many years, and therefore has low toxicity, and mammals (for example, humans, mice, rats, rabbits, dogs, Cats, cows, horses, pigs, monkeys, etc.).

  When administering or ingesting the composition and food according to the present invention, the dose or inoculation amount of the active ingredient according to the present invention is determined by the recipient, the age and weight of the recipient, symptoms, administration time, dosage form, administration method, drug It can be determined depending on the combination. For example, when the active ingredient according to the present invention is orally administered as a pharmaceutical, the amount of the protein per 1 adult (in terms of body weight) in the order of μg to mg is divided into 1 or several daily dosage units. be able to.

  The present invention will be described in detail by the following examples, but the present invention is not limited thereto.

Example 1: Extraction of RNA Japonica rice (variety: Koshihikari) seeds with 2,4-dichlorophenoxyacetic acid (2 mg / l; 2,4-D), sucrose (30 g / l) and agarose (8 g / l) Callus was induced by culturing at 25 ° C. in the dark using the DKN medium containing [Breed Sci. (2000) 50: 197]. The induced callus was cultured under light irradiation (300 lx) in a liquid DKN medium containing 2,4-D (1 mg / l) and sucrose (30 g / l) [Jpn. J. Breed (1988) 38 ( Suppl. 1): 78] and used for the experiment.
Total RNA was prepared from the rice callus derived from seeds using ISOGEN (manufactured by Nippon Gene).

Example 2a: Isolation of cDNA Encoding Oryzasistatin-I Using RNA extracted from rice callus, cDNA encoding oryzasistatin-I was obtained by PCR cloning. For the amplification reaction, TaKaRa One Step RNA PCR Kit (manufactured by Takara Bio Inc.) was used. The primers used for cloning of oryzasistatin-I cDNA were 5′-ATGTCGAGCGACGGAGGGCC-3 ′ (SEQ ID NO: 4) and 5′-GATGGGCCTTAGGCATTTGC-3 ′ (SEQ ID NO: 5). The PCR amplification product was subcloned into pCR2.1 using TA cloning Kit (Invitrogen).
In addition, the DNA sequencer (Model 373A; manufactured by Applied Biosystems) was used for the determination of the nucleotide sequence.

Example 2b: 5′-ATGGCCGAGGAGGCGCAGAG-3 ′ (SEQ ID NO: 6) and 5′-CTATGTACGTTTAGGCGGTG- in place of those shown in SEQ ID NOs: 4 and 5 were used for the isolation and cloning of cDNA encoding oryzastatin-III. A cDNA encoding oryzasistatin-III was isolated in the same manner as in Example 2a except that 3 ′ (SEQ ID NO: 7) was used.

Example 3: Construction of expression plasmids for oryzastatin-I and oryzastatin-III Oryzasistatin-I and oryzasistatin-III have a glutathione S-transferase (GST) sequence at the N-terminus, It was expressed as a fusion protein with a cleavage site by Factor Xa in between. A Factor Xa cleavage site (Ile-Glu-Gly-Arg) (SEQ ID NO: 8) was added immediately above the translation signals of oryzasistatin-I and oryzasistatin-III using a PCR reaction. The primers used for this purpose were 5′-ATCGAAGGTCGTATGTCGAGCGACGGAGGGCC-3 ′ (SEQ ID NO: 9) and 5′-GATGGGCCTTAGGCATTTGC-3 ′ (SEQ ID NO: 10) for oryzastatin-I, and 5′-ATCGAAGGTCGTATGTCGAGCGACGGAGGGCC-3 ′ (SEQ ID NO: 10). They were GGATCCATCGAAGGTCGTATGGCCGAGGAGGCGCAGCA-3 ′ (SEQ ID NO: 11) and 5′-TTAGGCGGTGGCGTCGTCGAGGGGCTTGAAATCC-3 ′ (SEQ ID NO: 12). The PCR amplification product was subcloned into pCR2.1 using TA cloning Kit (Invitrogen). After cleaving with a restriction enzyme, the oryzasistatin-I sequence added with Factor Xa recognition site is pGEX-4T-2 (Amersham Biosciences), and the oryzastatin-III sequence added with Factor Xa recognition site is pGEX-6P-2 ( Each of Amersham Biosciences), downstream of the GST coding region.

Example 4: Construction of an expression plasmid encoding oryzasistatin-II
Oryzasistatin-II cDNA with a Factor Xa recognition site added to the N-terminus was synthesized by PCR reaction using oryzasistatin-III cDNA as a template. The primers used for this were 5′-GGATCCATCGAAGGTCGTATGGCCGAGGAGGCGCAGAGCCACGCGCGTGAAGGTGGGCGGCATCCACGACAGCCGGCCGGGCGCGAGA-3 ′ (SEQ ID NO: 13) and 5′-TTAGGCGGTGGCGTCGTCGAGGGGCTTGAAATCC-3 ′ (SEQ ID NO: 14). The PCR amplified fragment was subcloned into pCR2.1 using TA cloning Kit (Invitrogen). After cleaving with a restriction enzyme, the oryzasistatin-II sequence added with Factor Xa recognition site was inserted downstream of the GST coding region of pGEX-6P-2 (Amersham Biosciences).

Example 5: Preparation of Recombinant Oryzasistatin-I, Oryzastatin-II, and Oryzasistatin-III E. coli BL21 strain (manufactured by Amersham Biosciences) into which GST-oryzastatin (-I / -II / -III) expression plasmid was introduced ) Was cultured at 37 ° C. using LB medium containing 100 μg / ml ampicillin. Three hours after the start of the culture, IPTG (Isopropyl-1-thio-beta-D-galactoside) was added to a concentration of 1.0 mM, and the culture was further performed for 6 hours. After completion of the culture, the collected cells are suspended in PBS (10 mM Na ₂ HPO ₄ /1.8 mM KH ₂ PO ₄ -140 mM NaCl-2.7 mM KCl) containing 1% Triton X-100 and subjected to ultrasonic treatment. Destroyed the cells. Subsequently, the solid content was removed by centrifugation, and then the fusion protein was affinity purified from the microbial cell disruption solution using a GSTRap HP column (manufactured by Amersham Biosciences). After collecting the fraction containing the fusion protein, it was dialyzed with 50 mM Tris-HCl (pH 8.0) -100 mM NaCl-5 mM CaCl ₂ and further Factor Xa (manufactured by Novagen) to a concentration of 5 U / ml. Was added, and the cleavage reaction was performed at 25 ° C. for 2 to 3 hours. After adsorbing and removing Factor Xa from the reaction solution using a HiTrap Benzamidine FF column (Amersham Biosciences), GST cleaved by GSTRap HP column chromatography was removed to obtain oryzasistatin-I, oryzatistatin-II, And a purified preparation of oryzasistatin-III was obtained.

Evaluation test: 0.1 M 2- (N-morpholino) ethanesulfonic acid-1 mM EDTA-4 mM dithiothreitol-0.05% Briji 35 was used as a buffer for measurement of cathepsin K inhibitory activity . To this, 11.6 nM recombinant human cathepsin K (manufactured by Calbiochem) and a predetermined concentration of oryzasistatin-I, oryzasistatin-II, or oryzasistatin-III are added and preincubated at 40 ° C. for 5 minutes. It was. Thereafter, Z-Phe-Arg-MCA (manufactured by Peptide Institute, Inc.) serving as an enzyme substrate was added to a concentration of 50 μM to initiate the enzyme reaction. Using a fluorescence spectrophotometer (Shimadzu RF-5300PC (manufactured by Shimadzu Corporation), measurement conditions: excitation wavelength 380 nm, absorption wavelength 440 nm), the increase in fluorescence intensity accompanying the progress of the reaction is monitored to The thickness was evaluated by the amount of AMC (7-amino-4-methyl-coumarin) released from the substrate. Enzyme activity 1U was defined as the amount of inhibitor that inhibits AMC release at 1 μmol / min.

The results were as shown in FIGS. FIG. 1 relates to oryzasistatin-I, FIG. 2 relates to oryzastatin-II, and FIG. 3 relates to oryzastatin-III.
From the results, these proteins strongly inhibited the activity of cathepsin K at a low concentration. Specific activities of enzyme inhibition were 7.40 U / mg for oryzastatin-I, 8.57 mU / mg for oryzastatin-II, and 1.98 U / mg for oryzastatin-III.

It is a graph which shows the measurement result of the cathepsin K inhibitory activity of oryzasistatin-I. It is a graph which shows the measurement result of the cathepsin K inhibitory activity of oryzasistatin-II. It is a graph which shows the measurement result of the cathepsin K inhibitory activity of oryzasistatin-III.

Claims (20)

  A cathepsin K inhibitor comprising a protein contained in a grass family plant as an active ingredient. The cathepsin K inhibitor according to claim 1, wherein the gramineous plant is rice ( Oryza sativa ).   The cathepsin K inhibitor according to claim 1 or 2, wherein the protein is oryzastatin-I, oryzastatin-II, or a partial peptide thereof.   The cathepsin K inhibitor according to claim 1 or 2, wherein the protein is oryzasistatin-III, which is a protein consisting of the amino acid sequence of SEQ ID NO: 3, or a partial peptide thereof. The cathepsin K inhibitor according to claim 1 or 2, wherein the protein is selected from the group consisting of the following (a) to (d):
(A) a protein comprising an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 3,
(B) a partial peptide consisting of a part of the amino acid sequence of (a), having a cathepsin K inhibitory activity,
(C) a protein comprising an amino acid sequence having at least 90% homology with the protein comprising the amino acid sequence of (a) and having cathepsin K inhibitory activity, and (d) the above (a) A protein comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted, added or inserted, and having cathepsin K inhibitory activity.   A composition comprising the protein according to any one of claims 1 to 5 as an active ingredient, which is used for treatment, prevention or amelioration of a disease or condition that can be treated, prevented or ameliorated by inhibiting cathepsin K.   7. The composition of claim 6, wherein the disease or condition is a disease or condition that can be induced by bone loss or increased bone resorption.   The composition according to claim 6 or 7, wherein the disease or condition is osteoporosis, osteoarthritis, rheumatoid arthritis, or a condition related thereto.   9. A composition according to any one of claims 6 to 8 provided in pharmaceutical form.   The composition according to any one of claims 6 to 8, which is provided in the form of food.   A food comprising an effective amount of the protein according to any one of claims 1 to 5, which is used for treatment, prevention or improvement of a disease or condition that can be treated, prevented or ameliorated by inhibiting cathepsin K. Food.   A food comprising an effective amount of the protein according to any one of claims 1 to 5, wherein the food is used for cathepsin K inhibition.   A food comprising an effective amount of the protein according to any one of claims 1 to 5, wherein a function of treating, preventing or ameliorating a disease or condition that can be treated, prevented or ameliorated by inhibiting cathepsin K is displayed. Food   A food comprising an effective amount of the protein according to any one of claims 1 to 5, wherein the food has an ability to inhibit cathepsin K.   The food according to claim 11 or 13, wherein the disease or condition is a disease or condition that can be induced by bone loss or increased bone resorption.   The food according to claim 11 or 13, wherein the disease or condition is osteoporosis, osteoarthritis, rheumatoid arthritis, or a condition related thereto.   The food according to any one of claims 13 to 16, wherein the indication of the function is any one of a main body, a container, a packaging, a manual, a package insert, or a promotional material.   The food according to any one of claims 11 to 17, which is a health food, a functional food, a food for specified health use, a dietary supplement, a food with a disease risk reduction label, or a food for a sick person. A protein selected from the group consisting of the following (a ′) to (d ′):
(A ′) a protein comprising an amino acid sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 3;
(B ′) a partial peptide consisting of a part of the amino acid sequence of (a ′), which has a cathepsin K inhibitory activity,
(C ′) a protein comprising an amino acid sequence having at least 90% homology with the protein comprising the amino acid sequence of (a ′) and having cathepsin K inhibitory activity, and (d ′) A protein comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted, added or inserted in the amino acid sequence of (a ′) and having cathepsin K inhibitory activity.   A method for inhibiting cathepsin K, comprising administering an effective amount of the protein according to any one of claims 1 to 5 to a mammal.

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