KR20100017708A - Novel antiphlogistic-analgesic agent - Google Patents

Abstract

The object is to provide a novel antiphlogistic-analgesic agent which is highly safe and can be administered over a long period. A study is made on the antiphlogistic-analgesic effect of α-lactalbumin which is one of natural substances contained in foods, and it is found that α-lactalbumin exhibits a cyclooxygenase-2 (COX-2) inhibition activity and a phospholipase-A2 inhibition activity. In thetest using a carrageenin food edema model, an adjuvant arthritis model and an acetic acid writhing test, it is also found that α-lactalbumin exhibits an anti-inflammatory effect. Further, it is also found that α-lactalbumin inhibits COX-2 more selectively compared to COX-1. Therefore, α-lactalbumin can be used as an antiphlogistic-analgesic agent having few adverse side effects.

Description

Novel antiphlogistic-analgesic agent

The present invention relates to a food and pharmaceutical composition having an anti-inflammatory action containing α-lactoalbumin.

Recently, as a medicine prescribed daily for anti-inflammatory pain, dozens of synthetic compounds such as aspirin, indomethacin, ibuprofen, diclofenac, collectively referred to as nonsteroidal anti-inflammatory drugs, and synthetic glucocorticoid hormone ).

Aspirin inhibits the activity of prostaglandin (PG) synthase "cyclooxygenase (COX)". PG decreases due to the decrease in COX activity, the activity of pain-causing substances (bradykinin) is suppressed, and the analgesic action of aspirin is expressed. Like aspirin, drugs that exert an anti-inflammatory analgesic effect on COX are called "non-steroidal anti-inflammatory drugs (NSAIDs)" to distinguish them from steroids with different mechanisms of action.

NSAIDs have defects that are susceptible to gastrointestinal ulcers. The main reason is that NSAIDs reduce PG (PGE ₂ , PGI ₂ ) which acts on vasodilation and mucosal protection. In addition, by suppressing COX activity, the activity of lipoxygenase is enhanced to increase leukotriene, and the secretion of gastric juice is reduced. At the same time, free radicals that destroy the gut mucosa increase, resulting in ulcers.

Prodrug types such as loxoprofen have been developed for the purpose of reducing such side effects of NSAIDs. However, the mechanism of action of the prodrug type is the same as that of conventional NSAIDs, and there is a limit to the side effect reduction effect.

The elimination of digestive disorders by NSAIDs has long been a challenge in the development of anti-inflammatory analgesics. The light on this challenge is the discovery of a new COX. COX-2, discovered in 1991, rapidly increases when trauma or inflammation occurs. On the other hand, the types of prostaglandins (PGE ₂ , PGI ₂ ) that existed in the body and act on the protection of the gastric mucosa were classified as COX-1. Accordingly, the substance selectively inhibiting the activity of COX-2 may be an anti-inflammatory analgesic with less side effects.

Side effects of NSAIDs include gastrointestinal disorders, nephropathy, liver disorders, skin rashes, and the like, as well as causing asthma attacks. Like conventional non-selective NSAIDs, when the action of COX-1 is suppressed along with inhibition of COX-2, which is the main action, the activity of lipoxygenase increases because the metabolism of arachidonic acid is biased toward leukotriene production. Leukotriene, which constricts neutrophils, increases, leading to asthma. Asthma by anti-inflammatory analgesics is called "aspirin asthma," but there is a risk of causing aspirin asthma not only in aspirin, but also in conventional NSAIDs. By the way, since the drug which inhibits only COX-2 does not increase leukotriene, it is said that there is little risk of causing asthma. In this respect too, expectations for COX-2 selective inhibitors are high. Etodolac, released in 1994, is used as an anti-inflammatory analgesic agent 10 times stronger in COX-2 and less digestive disorders. Meloxycam was also released in South Africa, France, the United Kingdom in 1996, and subsequently sold in Japan in 2001.

Some of the proteins in foods have anti-inflammatory action. For example, lactoferrin is known as a protein having anti-inflammatory action. Lactoferrin is an iron-bound glycoprotein of about 80,000 molecular weight, discovered in 1939. It is widely contained in breast milk, especially colostrum, and functions as an infection defense factor that prevents attacks of bacteria, viruses, and the like from the outside of the body. The safety of lactoferrin is highly appreciated, and lactoferrin extracted from milk is added to infant foods such as infant formula. Nowadays, lactoferrin's research on various diseases is drawing attention. Enhancement of biological defense against pathogenic microorganisms and viral infections in experimental animal models (e.g., Non-Patent Document 1), anti-inflammatory activity in various condition models, IgE production inhibitory effect (e.g., Non-patent Document 2) Preventive effects of carcinogenesis by chemical carcinogens (e.g., non-patent document 3), effects of inhibiting angiogenesis by cancer cells (e.g., non-patent document 4), etc. Is reported. It is also clear that lactoferrin enhances the analgesic effect of endogenous and exogenous opioids (for example, patent document 1).

In addition, lysozyme chloride is a basic protein having enzymaticity consisting of 129 amino acids of 18 species, and has an anti-inflammatory effect. As for the analgesic action, 50 to 300 mg / kg of mice was administered subcutaneously or orally, and analgesic action (hot plate method) was measured. As a result, no action was reported (Non-Patent Document 5).

Recently, whey and whey production fractions have been reported to have COX-2 inhibitory activity (Patent Document 7).

However, α-lactoalbumin is a globular protein having a molecular weight of 14,000, which accounts for about 25% of whey protein. α-lactoalbumin has high protein homology and high homology to lysozyme (Non-Patent Document 6). The properties of α-lactoalbumin are known to be calcium-binding proteins and high cystine contents. Further, the action of gastrointestinal motility (for example, Patent Literature 2), the prevention of small intestinal tract disorders or the promotion of repair, and the antiulcer effect (For example, nonpatent literature 7, patent document 5, and patent document 6) are known.

In addition, it has been reported that α-lactoalbumin suppresses the symptoms of premenstrual syndrome (Patent Document 8). However, the mechanism of action is not clear.

Patent Document 1: Japanese Patent No. 3806427

Patent Document 2: Japanese Patent No. 3756449

Patent Document 3: Japanese Patent Application Laid-Open No. 5-268879

Patent Document 4: Japanese Patent No. 2916047

Patent Document 5: Japanese Patent No. 3756449

Patent Document 6: Japanese Patent No. 3481931

Patent Document 7: Japanese Patent Publication No. 2007-505078

Patent Document 8: Japanese Patent Application Publication No. 2007-500755

Non Patent Literature 1: Lactoferrin; Structure, Function and Applications. Excerpta Medica Intern. Congress Series 1195 (2000), pp 377-381

[Non-Patent Document 2] Arthritis Rheum. 43 (2000) 2073-2080

[Non-Patent Document 3] Drug Metab. Pharmacokin. 19 (2004) 245-263

Non-Patent Document 4: Japan Medical Society October 2005 New Development of Lactoferrin Commercialization Research 10th Cancer Chemopreventive Effect and Lactoferrin Andunio

[Non-Patent Document 5] Drug Interview Form, Leaf Replep <Lysozyme Chloride Formulation>, Page 18

Non-Patent Document 6: Proc Natl Acad Sci U S A. 1992 July 1; 89 (13): 5887-5891.

Non-Patent Document 7: Biosci. Biotechnol. Biochem., 67 (3), 577-583, 2003

Disclosure of Invention

Problems to be Solved by the Invention

This invention is made | formed in view of the said situation, and an object of this invention is to provide the new anti-inflammatory analgesic agent which has few side effects, high safety, and can be ingested as a medicine or food-drinks for a long time.

Means to solve the problem

MEANS TO SOLVE THE PROBLEM This inventor earnestly researched in order to solve the said subject. In view of safety, the present inventors have carefully searched for substances having an anti-inflammatory effect among natural substances contained in foods. The anti-inflammatory analgesic effect of α-lactoalbumin was examined, and α-lactoalbumin showed cyclooxygenase (COX) -2 inhibitory activity and phospholipase A2 inhibitory activity in vitro. In addition, the validation of carrageenin foot edema model, adjuvant arthritis model, and in vivo by acetic acid writhing test can be used for acute inflammation, experimental chronic inflammation, and pain. It has become clear that it has a therapeutic effect. In addition, the inventors of the present invention examined the enzyme inhibitory activity of α-lactoalbumin, and it was clear that COX-2 was selectively inhibited over COX-1, and it was found that α-lactoalbumin can be used as an anti-inflammatory analgesic. The high safety of α-lactoalbumin is expected not only from COX-2 selective inhibitory action but also from long-term dietary experience. Conventionally, the anti-inflammatory analgesic effect of lactoferrin is known in the protein in plants, but the mechanism of action of the effect is not necessarily elucidated. Lysozyme chloride also has an anti-inflammatory effect, but no analgesic effect. On the other hand, the inventors of the present invention not only found α-lactoalbumin as a protein in foods having an anti-inflammatory analgesic effect, but also clarified that the effect was due to COX-2 selective inhibition. It is thought that COX-2 selective inhibitors could solve the side effect problem of conventional NSAIDs, and the above findings by the present inventors demonstrate that α-lactoalbumin has a very useful use as a novel anti-inflammatory analgesic. That is, this invention provides the following invention specifically about the novel use of (alpha)-lactoalbumin.

(1) a COX-2 selective inhibitor containing α-lactoalbumin as an active ingredient,

(2) anti-inflammatory analgesic containing α-lactoalbumin as an active ingredient,

(3) use of α-lactoalbumin for the manufacture of anti-inflammatory analgesics,

(4) the anti-inflammatory analgesic agent according to claim 2, which is an inflammatory disease treatment agent,

(5) the anti-inflammatory analgesic agent according to claim 4, wherein the inflammatory disease is arthritis or joint rheumatism,

(6) the anti-inflammatory analgesic agent according to claim 2, which is an analgesic disease treatment agent,

(7) the anti-inflammatory analgesic agent according to claim 6, wherein the painful disorder is dysmenorrhea,

(8) anti-inflammatory analgesic foods containing α-lactoalbumin as an active ingredient,

(9) a method of treating inflammatory diseases in which a therapeutically effective amount of α-lactoalbumin is administered to a mammal,

(10) A method of treating pain disorders in which a mammal is administered a therapeutically effective amount of α-lactoalbumin.

Effects of the Invention

Novel anti-inflammatory drugs have been provided by the present invention. Since the novel anti-inflammatory analgesic agent of the present invention selectively inhibits COX-2, side effects such as peptic ulcer and asthma are less likely to be ingested for a long time. α-lactoalbumin is included in milk of mammals as described above, and it is recognized that it is safe from a long food experience. In fact, the safety of α-lactoalbumin has been scientifically verified. For example, Patent Document 5 shows that the maximum dose of α-lactoalbumin, 2,000 mg / kg body weight, is the result of an acute toxicity test of α-lactoalbumin using rats. It has been reported that death was not confirmed by administration as in the blank test. Moreover, since it is a substance existing in food originally, it can be expected to process and distribute not only as a medicine but as a food-drink. In addition, since it can be obtained from relatively inexpensive raw materials such as milk, mass production is possible.

Lactoferrin in milk is industrially taken out by the separation technique from milk. The content of lactoferrin is somewhat different depending on the place of origin, the feed, etc., but in the case of domestic milk in Japan, it is usually a very small amount of 200 mg / kg. Therefore, milk sufficient to extract trace amounts of lactoferrin cannot be procured in Japan, and it is not produced in Japan at all, and all depend on imports.

The content of α-lactoalbumin in milk varies somewhat depending on the place of origin, feed, and the like, but is generally 1.2 g / kg for milk produced in Japan. It is richer than lactoferrin and is easy to industrially separate. Therefore, not only can it be secured securely, but also procurement cost becomes low.

Brief description of the drawings

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the change of foot volume in a carrageenan foot edema suppression test model. The mean value of 6 animals of 1 group + standard error was shown. Control is control, WPI is whey protein isolate, bLG is β-lactoglobulin, aLA is α-lactoalbumin, and LF is lactoferrin. *: vs. control, p <0.05 (Dunnet's multiple comparison test)

FIG. 2 is a graph showing the change of the foot volume in the adjuvant arthritis (treatment) model. The mean value of 6 animals of 1 group + standard error was shown. Vehicle is control, bLG is β-lactoglobulin, aLA is α-lactoalbumin, LF is lactoferrin, and Dicro is diclofenac. *, **, ***: vs. Vehicle, p <0.05, 0.01, 0.001 (Dunnet's multiple comparison test)

3 is a diagram showing the number of prostatic reactions in the adjuvant arthritis (pain) model. The mean value of 6 animals of 1 group + standard error was shown. Vehicle is control, bLG is β-lactoglobulin, aLA is α-lactoalbumin, LF is lactoferrin, and Dicro is diclofenac. *, **: vs. Vehicle, p <0.05, 0.01 (Dunnet's multiple comparison test)

It is a figure which shows the frequency | count of trouble operation in an acetic acid rising test model. The mean value of 6 animals of 1 group + standard error was shown. Vehicle represents control, WPI represents whey protein isolate, bLG represents β-lactoglobulin, aLA represents α-lactoalbumin, LF represents lactoferrin, and Dicro represents diclofenac. *, **: vs. Vehicle, p <0.05, 0.01 (Dunnet's multiple comparison test)

Fig. 5 is a diagram showing the results of the degree of pain of dysmenorrhea in the randomized double-blind crossover test of α-lactoalbumin for improvement of menstrual pain.

Fig. 6 is a diagram showing the results of the degree of the effect of α-lactoalbumin on dysmenorrhea in the randomized double-blind crossover test, which affects daily life due to dysmenorrhea.

Fig. 7 shows the results of the degree of use of analgesics in a randomized double-blind crossover test of α-lactoalbumin for improvement of menstrual pain.

Implement the invention  Best form for

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail. However, this invention is not limited to the following preferable embodiment, It can change freely within the range of this invention.

The present invention relates to a COX-2 selective inhibitor comprising α-lactoalbumin as an active ingredient. In the present invention, a COX-2 selective inhibitor means a substance having a stronger COX-2 inhibitory effect than a COX-1 inhibitory effect. The α-lactoalbumin of the present invention is not particularly derived, but is preferably derived from human and / or non-human mammals (cows, sheep, goats, horses, etc.), and more preferably human or bovine α-lactoalbumin. The amino acid sequence and base sequence of these (alpha)-lactoalbumin are already well-known, and are registered in the databases of EMBL, DDBJ, NCBI, etc. For example, bovine α-lactoalbumin is registered in DDBJ as ACCESSION No. J05147. The base sequence of bovine α-lactoalbumin is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. In the amino acid sequence set forth in SEQ ID NO: 2, positions 1 to 19 are signal peptides. In addition, in the amino acid sequence of SEQ ID NO: 2, the presence of a variant in which arginine (R) at position 29 is replaced with glutamine (Q) is known ("Milk Comprehensive Dictionary" p.35, Asakura Shoten). Α-lactoalbumin secreted in milk is a mature peptide with cleaved signal peptide. Α-lactoalbumin used in the examples herein is the above-mentioned mature peptide.

α-lactoalbumin is derived from the milk of mammals such as humans, cows, sheep and goats by precipitation of ammonium sulphate ("The Latest High Dairy Technology Manual", Dairy Technology Promotion Society, 122-125, 1975), Ultrafiltration (Patent Literature). 3) It can manufacture by well-known techniques, such as the ion exchange method (patent document 4). Although the content of α-lactoalbumin in milk varies slightly depending on the place of origin and feed, it is usually 1.2 g / kg for domestic milk, and a large amount of α-lactoalbumin can be prepared by using milk as a raw material. have. Moreover, you may synthesize | combine chemically based on an amino acid sequence and a nucleotide sequence, or you may manufacture by genetic engineering methods well known to those skilled in the art. More conveniently, commercially available α-lactoalbumin (for example, manufactured by Dabisco Co., Ltd.) can be used.

In the present invention, the α-lactoalbumin which can be used is not limited to the known amino acid sequence known as the natural form of the human or non-human mammal described above, and as long as it has COX-2 selective inhibitory activity, The polypeptide of the variant in which the mutation exists may be used. Examples of such polypeptides include naturally occurring variants, homologues, and artificial variants. Specifically, one or more amino acids are added, deleted, substituted, or inserted into the native α-lactoalbuminamino acid sequence. Polypeptides comprising a sequence comprising a sequence or a polypeptide encoded by a polynucleotide that hybridizes to a complementary strand of a polynucleotide encoding a native α-lactoalbumin under conditions that are stringent, and more specifically, And polypeptides having COX-2 selective inhibitory activity, including sequences in which one or a plurality of amino acids are added, deleted, substituted, or inserted into the amino acid sequence of SEQ ID NO: 2, and as another example, SEQ ID NO: Hybridized under conditions stringent to the complementary strand of the polynucleotide comprising the code region of the nucleotide sequence of 1; Li a nucleotide code, there may be mentioned a polypeptide having a selective COX-2 inhibitory activity.

Such variant polypeptides can be prepared by techniques known to those skilled in the art. For example, a total mRNA was prepared from animal cells expressing α-lactoalbumin such as mammary gland, and RT-PCR was obtained using a primer designed based on the nucleotide sequence of native α-lactoalbumin. Variant polypeptides can be obtained by introducing and expressing DNA into a host vector system with a suitable promoter. Alternatively, a cDNA library was prepared from animal cells, and a polynucleotide hybridized under conditions conducive to the probe was selected from the cDNA library using a probe prepared based on the nucleotide sequence of native α-lactoalbumin. Variant polypeptide can be obtained by introducing the polynucleotide into an appropriate host and expressing it. The promoter or host vector system can be appropriately selected by those skilled in the art from a known promoter or host vector system.

The stringent hybridization conditions can be appropriately selected by those skilled in the art. In one example, in a hybridization solution comprising 25% formamide, under more stringent conditions 50% formamide, 4 × SSC, 50 mM Hepes pH 7.0, 10 × Denhardt's solution, 20 μg / ml denatured salmon sperm DNA After prehybridization at 42 ° C overnight, a labeled probe is added and hybridization is performed by warming at 42 ° C overnight. The cleaning liquid and the temperature conditions in the subsequent washing are about 1 × SSC, 0.1% SDS, 37 ° C., and about 0.5 × SSC, 0.1% SDS, 42 ° C. as more stringent high-strength conditions. As strict high-strength conditions, it can carry out about "0.2 * SSC, 0.1% SDS, 65 degreeC". As the cleaning conditions for hybridization become more stringent as described above, isolation of polynucleotides having high homology with the probe sequence can be expected. However, the combination of the SSC, SDS, and temperature conditions are exemplary, and those skilled in the art will appreciate that or other factors (eg, probe concentration, probe length, hybridization reaction time, etc.) that determine the stringency of the hybridization. By suitably combining the above, it is possible to realize the same stringency as above.

Polypeptides encoded by polynucleotides isolated using such hybridization techniques usually have high homology to the above-described native polypeptide and amino acid sequence. High homology means homology of at least 40%, preferably at least 60%, more preferably at least 80%, more preferably at least 90%, even more preferably at least 95%.

Or by introducing site-specific or random site-specific mutations into the nucleotide sequence of native α-lactoalbumin by a cassette mutation method or PCR mutation method, and expressing the polynucleotide using an appropriate host vector system. Polypeptides can be obtained. Whether or not the polypeptide prepared as described above has COX-2 selective inhibitory activity can be confirmed by measuring the inhibitory activity of the polypeptide against COX-1 and 2 by a known method, for example, as described in Examples described later. This can be confirmed by the method. The amino acid sequence of the variant polypeptide having such COX-2 selective inhibitory activity has high homology to the native amino acid sequence, for example, the homology is 80% or more or 85% or more, preferably 90% or more. More preferably, it is 95% or more.

In the present embodiment, as is clear from the examples described later, in order to perfume the anti-inflammatory analgesic effect, it differs depending on the dosage form, symptoms, weight, etc., but the α-lactoalbumins, the active ingredient, per kg of body weight per day In terms of protein, about 0.002 mg to about 1,000 mg, preferably about 0.01 mg to about 200 mg, and most preferably about 0.2 mg to about 40 mg can be administered (ingested).

The dosage of α-lactoalbumin, the active ingredient of the anti-inflammatory analgesic agent of the present invention, is generally about 0.1 mg to about 50,000 mg per day, preferably about 0.5 mg to about 10,000 mg, and most preferably about 10 mg to about 2,000 mg can be administered at one time or in divided portions to a patient in need of treatment with the composition of the present invention before meals, after meals, between meals and / or before bedtime. Dosages can also be determined individually, depending on the age, weight, and purpose of the patient being administered.

Anti-inflammatory drugs of the present invention can be used in any form of medicines or food and drink. For example, it is expected to treat and / or prevent various inflammations and pains by directly administering them as medicines or directly ingesting them as special purpose foods or nutritional functional foods such as specific health foods. Various types of foods (milk, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crust, powdered formulas, formulas, bottled foods, nutrition), regardless of the form of liquids, pastes, solids, powders, etc. Food, frozen foods, processed foods, and other commercially available foods).

Foods containing an anti-inflammatory analgesic agent of the present invention can be used by mixing water, protein, sugar, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, spices and the like. As the protein, for example, whole milk powder, skim milk powder, partially skim milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin Animal and vegetable proteins such as lactoferrin, soy protein, egg protein, and hexaprotein, and their degradation products; And various milk derived components such as butter, whey mineral, cream, whey, nonprotein nitrogen, sialic acid, phospholipid, lactose and the like. You may contain peptides and amino acids, such as casein phosphopeptide, arginine, and lysine. Examples of the saccharides include sugars, processed starch (soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), plant fiber, and the like, in addition to dextrin. As a lipid, For example, animal fats and oils, such as these fractionation oil, hydrogenated oil, and transesterified oil; And vegetable oils such as palm oil, safflower oil, corn oil, rapeseed oil, palm oil, fractionated oils thereof, hydrogenated oils, and transesterified oils. As vitamins, for example, vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, folic acid Etc. As minerals, calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, etc. are mentioned, for example. Examples of the organic acid include malic acid, citric acid, lactic acid, tartaric acid, and the like. In the preparation of the food-drinks containing the anti-inflammatory analgesic agent of this invention, these may be synthetic | combination goods, any of natural-derived products, and / or food containing many may be used as a raw material. These components can be used in combination of 2 or more type. The form of the food may be either solid or liquid. Moreover, gel form etc. may be sufficient.

When the anti-inflammatory analgesic agent of the present invention is used as a medicine, it can be administered in various forms. As the form thereof, for example, oral administration by tablets, capsules, granules, powders, syrups, etc. may be used, but it is processed into preparations such as injections or liquids, and is used for cervical, intravenous, intradermal, muscle Other dosage forms such as injection may be used. These various formulations are known and commonly used in the field of preparation of pharmaceuticals such as excipients, binders, disintegrants, lubricants, odorants, dissolution aids, suspending agents, coating agents, solvents, isotonic agents, etc. according to conventional methods. Formulations may be formulated using an adjuvant. In addition, an appropriate amount of calcium may be included. In addition, an appropriate amount of vitamins, minerals, organic acids, sugars, amino acids, peptides and the like may be added.

Anti-inflammatory drugs of the present invention can be used for the treatment of inflammatory diseases. The anti-inflammatory analgesic agent of the present invention can be applied to, for example, arthritis, rheumatoid arthritis (running, chronic), ulcerative colitis, and clonal disease, but is not limited to these, and is generally applied to diseases involving inflammation. It is possible.

Types of arthritis include infectious, immune, and crystalline. Specifically, tuberculosis, gout, chronic arthritis rheumatoid, deformed arthrosis, acute rheumatism fever, psoriatic arthritis, acute joint rheumatism, Still's disease, Reiter's syndrome, ankylosing spondylitis are known. La Hiroo, Etsuro Ogata, Fumimaru Takaku, and Seichiro Darui, `` Latest Internal Medicine, Vol. 74 Joint Disease <Bone and Joint Disease 2>, published by Nakayama Shoten, p.19, 1995). As one of agents used in the treatment of these diseases, NSAIDs such as COX inhibitors are used.

Joint rheumatism is a chronic inflammatory disease in which multiple arthritis occurs. The exact cause is unclear, but three factors of constitution, immunological factors, and environmental factors are important. In the case of immunological factors, it is known that lymphocytes, which play an important role in immunity by secreting a substance called cytokine, have a strange effect. As a result, strange antibodies appear and bind to tissues of their joints to cause arthritis. Most of the initial symptoms are stiff joints during waking, pain and swelling of the joints. Joint pain is varied, such as spontaneous pain (stable), tenderness (壓痛), exercise pain (運動 痛). Other symptoms include swollen joints, fever, and redness. Arthritis is a synovial inflammation in which the synovial membrane is inflamed. It is symmetrically seen in the joints of the limbs. Frequently, the joints that get sick are deformed as they progress to the fingers, toes, hands, feet, elbows, knees, and shoulders. Once joint rheumatism develops, complete treatment is difficult and the patient will be with the disease for life.

Nowadays, medicines routinely prescribed to alleviate inflammatory pain in joints such as chronic joint rheumatism or inflammatory pain in bone junctions include dozens of synthetic compounds, such as aspirin, indomethacin, ibuprofen, and diclofenac, which are called NSAIDs. And synthetic glucocorticoids called steroids.

It is prostaglandin that mediates inflammatory pain, which is alleviated by inhibiting COX by NSAIDs to reduce / stop prostaglandin biosynthesis. However, as already described, their agents cause a high frequency of peptic ulcers after prolonged administration. Thus, methods for improving inflammatory pain in joints with a high degree of safety without causing peptic ulcers have long been explored. Recently, the topic of NSAIDs is a COX-2 inhibitor that specifically inhibits COX-2 induced at the inflammatory site and reduces peptic ulcer that was a side effect of NSAIDs (Hiroo Imura, Etsuro Ogata, Takaku Fumimaro). , Seichiro Darui, `` Latest Internal Medicine, Vol. 74 Joint Diseases <Bone and Joint Diseases 2>, Published by Nakayama Shoten, P.19, 1995, and Fukushima Masanori, `` Merck Manual Medical Information Family Edition, '' Nikkei BP G., p. 233-235, 1999).

In addition, the anti-inflammatory analgesic agent of the present invention can be effectively used for the relief of pain not accompanied by inflammation. There are two types of pain, acute pain and chronic pain. Acute pain (such as ankle sprains) is a warning sign of a wound or any disorder in the body. By responding to pain, it is a reaction to eliminate the cause and prevent deterioration. On the other hand, chronic pain means that the warning signal does not play a role, and pain does not go away with any treatment. Chronic pain includes neuropathic pain, nociceptive pain, or a combination thereof.

Cyclooxygenase (COX) is known as an enzyme of prostaglandin (PG) biosynthesis. At least three kinds of COX-1, COX-2, and COX-3 have been reported in COX. COX-1 exists physiologically, and is responsible for physiological actions such as stomach and kidney. The concentration does not change regardless of irritation or inflammation of LPS. COX-2, on the other hand, is involved in inflammation and tissue disorders. Although it is not expressed in a normal state, it is mainly produced newly from lactose cells by stimulation or inflammation of LPS, IL-1, etc., and is suppressed by dexamethasone. In other words, when COX-2 is selectively inhibited, side effects to the stomach and the kidney are less likely, and the effect of improving inflammation or tissue disorder can be expected.

Indications for COX inhibitors include: (1) chronic joint rheumatism, deformable arthrosis, fifty shoulders, dog's shoulder syndrome, low back pain, rheumatism and motor disorders such as hay fever and gout, and (2) postoperative and post-traumatic pain and cancer. Pain, dental pain, symptomatic neuralgia, stone pain, dysmenorrhea, painful disorders, (3) various infections, malignant tumors, fever, such as collagen disease, (4) cerebral infarction, transient ischemic attack, ischemic heart disease Indications that utilize antithrombotic or antiplatelet actions such as Kawasaki disease, proteinuria, and cancer metastasis, (5) Endothelial shock, arteriovascular patency, hypotension, Bartter syndrome, male infertility, immunosuppression, and strengthening immunotherapy mainly Indications due to the action are known (Hiroo Imura, Etsuro Ogata, Fumimaru Takaku, Seichiro Darui, `` Latest Internal Medicine, Volume 74 Joint Disease <Bone and Joint Diseases 2> '', Nakaya) Shoten issue, p.77-78, 1995 years). Therefore, the anti-inflammatory analgesic agent of the present invention having COX-2 inhibitory activity is not limited to the overall disease accompanied by inflammation, and can be applied to the various diseases described above.

In addition, all the prior art documents referred to in this specification are integrated in this specification as a reference.

Hereinafter, although an Example is given and described about this invention, this invention is not limited by this.

Example 1 Carrageenan Foot Edema Inhibition Test, Action against Acute Inflammation

This example demonstrated the inhibitory effect on whey protein isolate (WPI) and carrageenan-induced paw edema of various proteins contained in WPI. WPI is a process in which whey is separated by microfiltration (MF), ultrafiltration (UF), cross-polo microfiltration (CFM), ion exchange, and the like to separate proteins and increase protein concentration.

(Way)

Various test substances were orally administered using Wister rats (male, 6-week-old, six-group, five-group) that had undergone one or more weeks of purification. As a test substance, 1 g / kg of whey protein isolate (WPI, cow derived, manufactured by Davisco Foods International, Inc.) dissolved in physiological saline [WPI group], α-lactoalbumin (α-LA, cow derived, Davisco Foods International , Inc.) 250 mg / kg (25 w / w% of WPI) [aLA group], β-lactoglobulin (β-LG, derived from Bovine, Davisco Foods International, Inc.) 500 mg / kg (WPI 50 w / w%) [bLG group], lactoferrin (LF, cow derived, manufactured by Murray Goulburn Ingredients) 10 mg / kg (1 w / w% of WPI) [LF group], and physiological saline [control] The dose of was administered at a dose of 4 mL / kg. One hour after oral administration of the test substance, 1 w / w% carrageenan solution was administered 0.1 mL intradermally to the left foot. Foot volume was measured at 3 hours after carrageenan administration, and its anti-inflammatory action was evaluated. In addition, in the following Example, WPI, (alpha) -LA, (beta) -LG, and LF used the same thing as what is shown here.

(result)

Among the WPIs, α-lactoalbumin (25 w / w% of WPI), β-lactoglobulin (50 w / w% of WPI), and lactoferrin (1 w / w% of WPI) are present. This time, in order to examine the active ingredient in WPI, it administered according to the content rate. As a result, only carbo-albumin was shown to inhibit carrageenan edema. From these results, it was found that the inhibitory effect of carpiginous paw edema of WPI was contributed by α-lactoalbumin (FIG. 1).

Example 2 (Adjuvant Arthritis (Treatment) Model, Action on Subacute Inflammation (Experimental Chronic Inflammation)

In this example, a rat adjuvant arthritis model, which is an animal model of rheumatoid arthritis, was produced, and the therapeutic effect of various proteins contained in the WPI was verified.

(Way)

After the end of one or more weeks of purification, adjuvant arthritis was caused by subcutaneous injection of 0.1 mL of Freund's complete adjuvant into the left paw of the Wister rats (male, 6 week old). Freund's complete adjuvant contains, in 10 mL, 8.5 mL of liquid paraffin, 1.5 mL of surfactant Span 80, and 7.5 mg of humanoid Mycobacterium tuberculosis H37Rv. After 14 days from adjuvant administration, rats with sufficient arthritis were selected and grouped based on the paw volume of the left foot (group 1, group 6, group 5).

Test substances dissolved in physiological saline (α-lactoalbumin [aLA group], β-lactoglobulin [bLG group], lactoferrin [LF group]) were doses of 300 mg / kg, respectively, which are nonsteroidal anti-inflammatory drugs (NSAIDs). Diclofenac (manufactured by Wako Pure Chemical Industries, Ltd.) [positive control] was administered orally at a dose of 50 mg / kg for 3 days from adjuvant to 14 to 16 days at a dose of 4 mL / kg. In addition, the same dose of physiological saline was administered orally for three consecutive days from the adjuvant administration to 14-16 days.

On the 14th day immediately after administration of the adjuvant, 15 and 17 days, the volume of the foot of the left foot was measured to investigate the therapeutic effect.

(result)

For edema of the left foot, which received the adjuvant, α-lactoalbumin inhibited the swelling of the edema. In addition, in the administration of lactoferrin, the paw volume decreased, and a therapeutic effect was obtained (FIG. 2).

Example 3 (Adjuvant Arthritis (Pain) Model, Action on Chronic Pain)

After the end of one or more weeks of acclimation, adjuvant arthritis was induced by subcutaneous injection of 0.1 mL of Freund's complete adjuvant as in Example 2, to the left foot of the Wister rat (male, 6 week old). Sixteen days after the adjuvant administration, rats with sufficient arthritis were selected and grouped based on the paw volume of the left paw (6 groups, 6 groups).

Test substances dissolved in physiological saline (α-lactoalbumin [aLA group], β-lactoglobulin [bLG group], lactoferrin [LF group]) were doses of 300 mg / kg, respectively, and diclofenac [positive control] It was orally administered at a dose of 50 mg / kg and a dose of 4 ml / kg. One hour after administration of the test substance, the presence or absence of a prognosis response to flexion extension stimulation pain of the left hind limb was measured. The number of propositions for 10 times of flexion was quantified to evaluate the effect of suppressing pain.

(result)

In single administration of α-lactoalbumin and single administration of lactoferrin, an inhibitory effect of arthritis pain was obtained (FIG. 3).

Example 4 Acetic Acid Rising Test, Action on Pain

In this example, the therapeutic effect of various proteins contained in WPI against acetic acid-induced pain was verified.

Whey protein isolates (WPI) [WPI] and [alpha] -lactoalbumin [αLA group] dissolved in physiological saline using mice that had been purified for at least one week (Woong, 6-week-old, group 6, group 5). , β-lactoglobulin [bLG group] and lactoferrin [LF group] at a dose of 300 mg / kg, respectively, and the anti-inflammatory analgesic diclofenac [Dicro group] at a dose of 50 mg / kg, 4 ml / kg. The dose was orally administered. In addition, as a vehicle group, the same amount of saline was orally administered. One hour after oral administration of the test substance, 0.2 mL of 0.6 v / v% acetic acid solution was administered intraperitoneally. The pain suppression effect was evaluated by numerically counting the number of distress movements for 20 minutes from 10 minutes to 30 minutes after acetic acid administration.

(result)

The results are shown in FIG. There were significant analgesic effects on α-lactoalbumin and lactoferrin.

Example 5 (Enzyme Inhibition Experiment)

In this example, the inhibitory activity against various enzymes of inflammation of the test substance (α-lactoalbumin, WPI) was verified.

(Way)

(1) Activity measurement using the enzyme standard of cyclooxygenase-1 (COX-1) (n = 2)

Enzyme samples of various concentrations of test substance (α-lactoalbumin or WPI) and cyclooxygenase-1 (EC 1.14.99.1, derived from human platelets) were prepared by HBSS (Hanks' Balanced Salt Solutions, 15 mM HEPES; pH 7.4). After preincubation at 37 ° C. for 15 minutes, arachidonic acid was added to a final concentration of 100 μM and reacted at 37 ° C. for 15 minutes. After the reaction, the concentration of PGE ₂ was measured by an enzyme immunoassay (EIA). In addition, no test substance (α-lactoalbumin or WPI) was added as a control. Using the mean value of the control value as the enzyme inhibition rate of 100%, the enzyme inhibition rate of α-lactoalbumin and WPI was calculated, and the 50% inhibition concentration was set as the IC ₅₀ value.

(2) Activity measurement using enzyme standard of cyclooxygenase-2 (COX-2) (n = 2)

Enzyme samples of various concentrations of test substance (α-lactoalbumin or WPI) and cyclooxygenase-2 (EC 1.14.99.1, human recombination; Sf21 insect cells) were prepared using 1 mM glutathione, 1 μM hematin, 500 μM. After preincubation at 37 ° C. for 15 minutes in 100 mM Tris-HCl buffer containing phenol (pH 7.7), arachidonic acid was added to a final concentration of 0.3 μM and allowed to react at 37 ° C. for 5 minutes. After the reaction, the concentration of PGE ₂ was measured by EIA. In addition, no test substance (α-lactoalbumin or WPI) was added as a control. Using the mean value of the control value as the enzyme inhibition rate of 100%, the enzyme inhibition rate of α-lactoalbumin and WPI was calculated, and the 50% inhibition concentration was set as the IC ₅₀ value.

(3) Activity measurement using enzyme standard of inducible nitric oxide synthase (iNOS) (n = 2)

Enzyme samples of various concentrations of the test substance (α-lactoalbumin or WPI) and inducible nitric oxide synthase (EC 1.14.13.39, derived from mouse macrophages) were prepared using 4 mM NADPH (reduced nicotinamide adenine dinucleotide), 8 μM ( 6R) -5,6,7,8-tetrahydro-L-biopterin, 8 μM FAD, 6 mM pre-incubation in 100 mM Tris-HCl buffer (pH 7.9) containing 6 mM DTT (Dithiothreitol) for 15 minutes After that, arginine was added to a final concentration of 100 μM and reacted at 37 ° C. for 2 hours. After the reaction, NO ^2- was quantified by spectrophotometry. In addition, no test substance (α-lactoalbumin or WPI) was added as a control. The enzyme inhibition rate of α-lactoalbumin and WPI was calculated by making the average value of the quantitative value of the control group 100% enzyme inhibition rate, and 50% inhibition concentration was made into IC ₅₀ value.

(4) Determination of activity using enzyme standard of phospholipase A2 (PLA2) (n = 2)

Enzymes of various concentrations of test substance (α-lactoalbumin or WPI) and phospholipase A2-I (EC 3.1.1.4, derived from porcine pancreas) or phospholipase A2-II (EC 3.1.1.4, derived from venomous rattlesnake) The product was preincubated in 0.1 M Glycine-NaOH buffer containing 20 μM EDTA (pH 9.0) for 15 minutes at 37 ° C., followed by 1-palmitoyl-2- [1- ¹⁴ C] oleoyl-L-3-phosphatidylcholine Was added to a final concentration of 0.03 μCi and reacted for 5 minutes at 37 ° C. After removal of unreacted 1-palmitoyl-2- [1- ¹⁴ C] oleoyl-L-3-phosphatidylcholine, the radioactivity of the oleate fractions was measured with a liquid scintillation counter. In addition, no control substance (α-lactoalbumin or WPI) was added as a control. The enzyme inhibition rate of α-lactoalbumin and WPI was calculated using the average value of the radioactivity (Ci) of the control group as the enzyme inhibition rate of 100%, and the 50% inhibition concentration was set as the IC ₅₀ value.

(result)

The results are shown in Table 1 and Table 2. α-lactoalbumin showed high activity in cyclooxygenase-2, phospholipase A2-I, and phospholipase A2-II. In particular, selective high activity could be found for cyclooxygenase-2. It was also found that α-lactoalbumin had high cyclooxygenase-2 activity even when compared to WPI.

Example 6 (Human Test for Dysmenorrhea)

In this example, the improvement effect of α-lactoalbumin on dysmenorrhea was verified by a randomized double blind crossover test.

(Subject)

A preliminary survey was conducted on 70 women who applied for cooperation in posting a request for cooperation, and the menstrual cycle stabilized at 28 days ± 3 days, and a magnetic questionnaire about recent menstruation (Table 3: Pain in menstruation and dysmenorrhea) 30 women (degrees of influence on daily life, use of painkiller) 30 or more women (more than pain degree of dysmenorrhea, the sum of menstrual pain score (degree of influence on daily life by dysmenorrhea, use of painkiller) 2 or more 22-50 years old, average 36.4 years old) were selected and were the subjects.

(Subject)

Tablets A and B with identical appearances were prepared. One is an α-lactoalbumin tablet (containing 150 mg of α-lactoalbumin in one tablet), and the other contains no α-lactoalbumin at all (placebo tablet). Both the subject and the evaluator started a test in the state which hidden which tablet was the α-lactoalbumin tablet until the evaluation completion.

(Ingestion method)

Tests were conducted during the March-May cycle between September and December 2007. The menstrual cycle from the end of the first menstruation to the end of the next menstruation was placed as the first test subject. The menstrual cycle (from the end of menstruation to the end of the next menstruation) following the first subject intake period was set to rest without ingesting the subject. In addition, the menstrual cycle (from the end of menstruation to the end of the next menstruation) following the rest period was set as the second test subject intake.

Subjects were divided into two groups, group 1 (tablet A intake-rest-table B intake, n = 13) and group 2 (tablet B intake-rest-table A intake, n = 17). The order of intake of tablets and A, tablets and B, respectively, was set differently.

The test subject ingested two tablets (tablet A or tablet B) three times each morning, lunch and dinner.

In addition, the use of analgesics was freed before the start of the test and during the test period.

(Assessment Methods)

During one menstrual period prior to commencement of the study, subjects were asked to fill out a magnetic questionnaire (Table 3). In addition, between the subject intake period and the resting period, the subject was continuously recorded in the self-reported questionnaire once a day. The content of the magnetic questionnaire is about dysmenorrhea and QOL. The degree of menstrual pain, the effects on daily life, and the use of analgesics were recorded on the questionnaire during the menstrual period using the VRS (verbal rating scale), and the maximum value during each period was used for evaluation. . In addition, menstrual pain was evaluated as low back pain and lower abdominal pain.

In addition, the average value of the results of menstruation and rest periods before the start of the test was obtained, and this was taken as the value of the non-intake period.

(Statistical processing)

The mean value and standard error were obtained for each measured value, and the test of the significant difference was performed as a non-parametric method by using statistical software (Wilcoxson's sign rank sum test).

(result)

The VRS (verbal rating scale) value, measured for pain in menstrual pain, was significantly (p <0.01) lowered with the intake of α-lactoalbumin compared to the non-ingested group (FIG. 5).

The VRS (verbal rating scale) value for assessing the effect of daily dysmenorrhea (QOL) on dysmenorrhea tended to decrease (p <0.10) with the intake of α-lactoalbumin compared to the non-ingestion group (Fig. 6). ).

In terms of the use of analgesics, 21 patients (70%) took analgesics for menstrual pain before the start of the test, but decreased to 10 (33% of total) in the α-lactobumin tablets and placebo tablets. The VRS (verbal rating scale) value, which measured the use of analgesics, was found to be significantly lower in both tablets and intakes compared to the non-intake group (p <0.01) (FIG. 7).

From the above results, by ingesting 300 mg of α-lactoalbumin three times a day (morning, lunch and dinner), the use rate of analgesics was greatly reduced, and the effect of alleviating menstrual pain and improving QOL became clear.

Α-lactoalbumins, which are the active ingredient in the present invention, can be mass-produced from relatively inexpensive raw materials such as milk. Moreover, since there are few side effects, high safety, and it is possible to ingest over a long period of time, the anti-inflammatory analgesic effect can be enjoyed by mixing with pharmaceuticals or ingesting as a food. Not only this, but it is also possible to apply it to the use, such as manufacture of functional foods for the patient in a low nutrition state, since sufficient nutrition management is possible by daily administration or ingestion.

                         SEQUENCE LISTING <110> MEIJI DAIRIES CORPORATION   <120> A NOVEL ANTIINFLAMMATORY AND ANALGESIC AGENT <130> M1-A0704P <150> JP 2007-127286 <151> 2007-05-11 <160> 2 <170> PatentIn version 3.4 <210> 1 <211> 724 <212> DNA <213> Bos taurus <220> <221> CDS <222> (28) .. (456) <220> <221> sig_peptide (222) (28) .. (84) <220> <221> mat_peptide (222) (85) .. (453) <400> 1 atttcagaat cttggggggt aaccaaa atg atg tcc ttt gtc tct ctg ctc ctg 54                               Met Met Ser Phe Val Ser Leu Leu Leu                                               -15 gta ggc atc cta ttc cat gcc acc cag gct gaa cag tta aca aaa tgt 102 Val Gly Ile Leu Phe His Ala Thr Gln Ala Glu Gln Leu Thr Lys Cys -10 -5 -1 1 5 gag gtg ttc cgg gag ctg aaa gac ttg aag ggc tac gga ggt gtc agt 150 Glu Val Phe Arg Glu Leu Lys Asp Leu Lys Gly Tyr Gly Gly Val Ser             10 15 20 ttg cct gaa tgg gtc tgt acc acg ttt cat acc agt ggt tat gac aca 198 Leu Pro Glu Trp Val Cys Thr Thr Phe His Thr Ser Gly Tyr Asp Thr         25 30 35 caa gcc ata gta caa aac aat gac agc aca gaa tat gga ctc ttc cag 246 Gln Ala Ile Val Gln Asn Asn Asp Ser Thr Glu Tyr Gly Leu Phe Gln     40 45 50 ata aat aat aaa att tgg tgc aaa gac gac cag aac cct cac tca agc 294 Ile Asn Asn Lys Ile Trp Cys Lys Asp Asp Gln Asn Pro His Ser Ser 55 60 65 70 aac atc tgt aac atc tcc tgt gac aag ttc ctg gat gat gat ctt act 342 Asn Ile Cys Asn Ile Ser Cys Asp Lys Phe Leu Asp Asp Asp Leu Thr                 75 80 85 gat gac att atg tgt gtc aag aag att ctg gat aaa gta gga att aac 390 Asp Asp Ile Met Cys Val Lys Lys Ile Leu Asp Lys Val Gly Ile Asn             90 95 100 tac tgg ttg gcc cat aaa gca ctc tgt tct gag aag ctg gat cag tgg 438 Tyr Trp Leu Ala His Lys Ala Leu Cys Ser Glu Lys Leu Asp Gln Trp         105 110 115 ctc tgt gag aag ttg tgaacacctg ctgtctttgc tgcttctgtc ctctttctgt 493 Leu Cys Glu Lys Leu     120 tcctggaact cctctgcccc gtggctacct cgttttgctt ctttgtaccc ccttgaagct 553 aactcgtctc tgagccctgg gccctgtagt gacaatggac atgtaaggac taatctccag 613 gggtgcatga atggcgctct ggacttttga cccttsstcg atgtccctga tggcgctttt 673 aatgcaacag tacatattcc acttttgtcc cgaataaaaa gcctgatttt g 724 <210> 2 <211> 142 <212> PRT <213> Bos taurus <400> 2 Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe His Ala                 -15 -10 -5 Thr Gln Ala Glu Gln Leu Thr Lys Cys Glu Val Phe Arg Glu Leu Lys         -1 1 5 10 Asp Leu Lys Gly Tyr Gly Gly Val Ser Leu Pro Glu Trp Val Cys Thr     15 20 25 Thr Phe His Thr Ser Gly Tyr Asp Thr Gln Ala Ile Val Gln Asn Asn 30 35 40 45 Asp Ser Thr Glu Tyr Gly Leu Phe Gln Ile Asn Asn Lys Ile Trp Cys                 50 55 60 Lys Asp Asp Gln Asn Pro His Ser Ser Asn Ile Cys Asn Ile Ser Cys             65 70 75 Asp Lys Phe Leu Asp Asp Asp Leu Thr Asp Asp Ile Met Cys Val Lys         80 85 90 Lys Ile Leu Asp Lys Val Gly Ile Asn Tyr Trp Leu Ala His Lys Ala     95 100 105 Leu Cys Ser Glu Lys Leu Asp Gln Trp Leu Cys Glu Lys Leu 110 115 120  

Claims (10)

COX-2 selective inhibitor which uses alpha-lactoalbumin as an active ingredient. Anti-inflammatory analgesic containing alpha-lactoalbumin as an active ingredient. Use of α-lactoalbumin for the preparation of anti-inflammatory analgesics. The method of claim 2, Anti-inflammatory analgesic drug for the treatment of inflammatory diseases. The method of claim 4, wherein Anti-inflammatory drugs, in which the inflammatory disease is arthritis or joint rheumatism. The method of claim 2, Anti-inflammatory analgesic drug for the treatment of pain. The method of claim 6, Anti-inflammatory drugs that have painful menstruation. Anti-inflammatory analgesic foods containing α-lactoalbumin as an active ingredient. A method of treating inflammatory disease wherein a therapeutically effective amount of α-lactoalbumin is administered to a mammal. A method for treating painful pain, wherein a mammal is administered a therapeutically effective amount of α-lactoalbumin.

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