JP2013241418A - New antiphlogistic-analgesic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new highly safe nonsteroidal antiphlogistic-analgesic agent capable of being administered over a long period.SOLUTION: A cyclooxygenase-2 (COX-2) selective inhibitor, an antiphlogistic-analgesic agent, an inflammatory disorder (arthritis, articular rheumatism, and the like) therapeutic drug, painful disease (menstrual pain and the like) therapeutic drug, and antiphlogistic-analgesic food and drink using α-lactalbumin as an active ingredient are provided.

Description

  The present invention relates to foods and drinks having anti-inflammatory analgesic activity and pharmaceutical compositions containing α-lactalbumin.

  Today, dozens of synthetic compounds, such as aspirin, indomethacin, ibuprofen, and diclofenac, which are commonly called nonsteroidal anti-inflammatory analgesics, and synthetic glucos, commonly called steroids, are routinely prescribed for anti-inflammatory analgesia. There are corticoid hormones.

  Aspirin suppresses the activity of the prostaglandin (PG) synthase “cyclooxygenase (COX)”. Reduced COX activity reduces PG, suppresses the activity of pain-inducing substances (bradiginin), and develops aspirin analgesia. Drugs that exert anti-inflammatory analgesic effects by acting on COX as well as aspirin are classified as “non-steroidal anti-inflammatory drugs (NSAIDs)” to distinguish them from steroids with different mechanisms of action. is called.

NSAIDs have the disadvantage of causing gastrointestinal ulcers. The main cause is that NSAIDs reduce PG (PGE ₂ , PGI ₂ ), which acts for vasodilation and mucosal protection. In addition, suppression of COX activity increases lipoxygenase activity, increases leukotrienes, and decreases gastric juice secretion. At the same time, active oxygen that destroys the gastrointestinal mucosa increases and ulcers occur.

  In order to reduce such side effects of NSAIDs, prodrug types such as loxoprofen have been developed. However, the mechanism of action of the prodrug type is the same as that of conventional NSAIDs, and the side effect reduction effect is limited.

Resolving gastrointestinal disorders with NSAIDs has been a long-standing research subject in the development of anti-inflammatory analgesics. The discovery of a new COX shed light on this challenge. COX-2, discovered in 1991, increases rapidly when trauma or inflammation occurs. On the other hand, the type that produces prostaglandins (PGE ₂ , PGI ₂ ) that originally exist in the body and that protect the gastric mucosa was classified as COX-1. Therefore, a substance that selectively inhibits the activity of COX-2 can be an anti-inflammatory analgesic with few side effects.

  In addition to gastrointestinal disorders, NSAIDs have side effects such as kidney damage, liver damage, skin rash, and other serious side effects such as induction of 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, lipoxygenase activity increases because arachidonic acid metabolism is biased toward leukotriene production. Leukotriene, which causes bronchoconstriction, increases and asthma is induced. Asthma due to anti-inflammatory analgesics is called “aspirin asthma”, but there is a risk of inducing aspirin asthma not only in aspirin but in all conventional NSAIDs. However, drugs that only inhibit COX-2 do not increase leukotrienes, so it is said that there is less risk of inducing asthma. Also from this point, expectation for a COX-2 selective inhibitor is high. Etodolac, launched in 1994, is 10 times stronger on COX-2 and is used as an anti-inflammatory analgesic with relatively little digestive damage. Meloxicam was released in 1996 in South Africa, France, the UK, etc., and later in Japan in 2001.

  Some of the proteins contained in food have anti-inflammatory analgesic action. For example, lactoferrin is known as a protein having anti-inflammatory analgesic action. Lactoferrin is an iron-binding glycoprotein with a molecular weight of about 80,000 discovered in 1939. It is abundant in breast milk, especially colostrum, and functions as an infection defense factor that prevents attacks from bacteria and viruses from outside the body. The safety of lactoferrin is highly evaluated, and lactoferrin extracted from milk has been added to infant foods and drinks such as infant formula. In recent years, research on lactoferrin has attracted attention for various diseases. Strengthening of biological defenses against pathogenic microorganisms and virus infection in experimental animal models (for example, Non-Patent Document 1), anti-inflammatory action in various pathological models, IgE production inhibitory effect (for example, Non-Patent Document 2), carcinogenesis by chemical carcinogens A function of controlling various immune-related life phenomena, such as a prophylactic effect (for example, Non-Patent Document 3) and an effect of inhibiting angiogenesis by cancer cells (for example, Non-Patent Document 4) have been reported. It has also been clarified that lactoferrin enhances the analgesic effect of endogenous and exogenous opioids (for example, Patent Document 1).

  Lysozyme chloride is a basic protein having an enzymatic property composed of 129 amino acids of 18 types and has an anti-inflammatory effect. Regarding analgesic action, 50-300 mg / kg was administered subcutaneously or orally to mice, and the analgesic action (hot plate method) was measured, and as a result, no action was reported (Non-patent Document 5).

  In recent years, it has been reported that whey and whey production fractions have COX-2 inhibitory activity (Patent Document 7).

  By the way, α-lactalbumin is a globular protein having a molecular weight of 14,000, accounting for about 25% of whey protein. α-Lactalbumin is highly homologous in protein structure and gene sequence with lysozyme (Non-patent Document 6). It is known that α-lactalbumin is a calcium-binding protein and has a high cystine content. Further, it regulates gastrointestinal motility (for example, Patent Document 2), promotes prevention or repair of intestinal injury. An action and an anti-ulcer action (for example, Non-Patent Document 7, Patent Document 5, and Patent Document 6) are known.

  In addition, α-lactalbumin has been reported to suppress symptoms of premenstrual syndrome (Patent Document 8). However, its mechanism of action is not clear.

Japanese Patent No. 3806427 Japanese Patent No. 3756449 Japanese Patent Laid-Open No. 5-28879 Japanese Patent No. 2916047 Japanese Patent No. 3756449 Japanese Patent No. 3348311 Special Table 2007-505078 Special Table 2007-500755

Lactoferrin; Structure, Function and Applications.Excerpta Medica Intern.Congress Series 1195 (2000), pp377-381 Arthritis Rheum. 43 (2000) 2073-2080 Drug Metab. Pharmacokin. 19 (2004) 245-263 Japan Medical Society October 2005 New Development of Lactoferrin Practical Research 10th Cancer Chemopreventive Effect and Lactoferrin Kunio Ando Pharmaceutical Interview Form Riflap <Lysozyme Chloride Preparation> 18 Eisai Co., Ltd. Proc Natl Acad Sci U S A. 1992 July 1; 89 (13): 5887-5891. Biosci. Biotechnol. Biochem., 67 (3), 577-583, 2003

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a novel anti-inflammatory analgesic that has few side effects, is highly safe, and can be ingested as a medicine or food and drink for a long period of time. .

The present inventors have intensively studied to solve the above problems. In consideration of safety, the present inventors have searched carefully for substances having anti-inflammatory effects from natural substances contained in foods. When anti-inflammatory analgesic action was examined for α-lactalbumin, α-lactalbumin exhibited cyclooxygenase (COX) -2 inhibitory activity and phospholipase A2 inhibitory activity in vitro. In vivo verification using a carrageenan paw edema model, an adjuvant arthritis model, and the acetic acid-rising method revealed a therapeutic effect on acute inflammation, experimental chronic inflammation, and pain. Furthermore, when the present inventors examined the enzyme inhibitory activity of α-lactalbumin, it was found that it selectively inhibits COX-2 over COX-1, and α-lactalbumin was used as an anti-inflammatory analgesic. I found out. The high safety of α-lactalbumin is not only expected from the selective inhibitory action of COX-2, but can be said to be ensured by a long dietary experience. Conventionally, anti-inflammatory analgesic effects of lactoferrin have been known for proteins in food, but the mechanism of action of such effects has not necessarily been elucidated. Although lysozyme chloride has an anti-inflammatory effect, it has no analgesic effect. On the other hand, the inventor of the present application not only discovered α-lactalbumin as a protein in foods having anti-inflammatory analgesic effect, but also clarified that the effect is due to COX-2 selective inhibitory action. COX-2 selective inhibitors are thought to solve the side effects of conventional NSAIDs, and the above findings by the present inventors indicate that α-lactalbumin has a very useful use as a novel anti-inflammatory analgesic. Prove that. That is, the present invention relates to a novel use of α-lactalbumin, and specifically provides the following inventions.
(1) a COX-2 selective inhibitor comprising α-lactalbumin as an active ingredient,
(2) an anti-inflammatory analgesic comprising α-lactalbumin as an active ingredient,
(3) Use of α-lactalbumin for the manufacture of anti-inflammatory analgesics,
(4) An anti-inflammatory analgesic according to (2), which is a therapeutic agent for inflammatory diseases,
(5) The anti-inflammatory analgesic according to (4), wherein the inflammatory disease is arthritis or rheumatoid arthritis,
(6) The anti-inflammatory analgesic according to (2), which is a therapeutic agent for painful diseases,
(7) The anti-inflammatory analgesic according to (6), wherein the painful disease is menstrual pain,
(8) Anti-inflammatory analgesic food or drink comprising α-lactalbumin as an active ingredient,
(9) A method for treating an inflammatory disease, comprising administering a therapeutically effective amount of α-lactalbumin to a mammal,
(10) A method for treating painful diseases, wherein a therapeutically effective amount of α-lactalbumin is administered to a mammal.

  The present invention provides a novel anti-inflammatory analgesic. Since the novel anti-inflammatory analgesic of the present invention selectively inhibits COX-2, side effects such as peptic ulcer and asthma are few, and it can be taken for a long time. As described above, α-lactalbumin is contained in the milk of mammals and has been recognized as safe from a long dietary experience. In fact, the safety of α-lactalbumin has been scientifically verified. For example, Patent Document 5 discloses that the maximum dose of 2,000 mg / kg is the result of an acute toxicity test of α-lactalbumin using rats. As with the blank test, no deaths were reported after administration of body weight α-lactalbumin. In addition, since it is a substance originally present in food, it can be expected to be processed and distributed not only as a pharmaceutical product but also as a food and drink. Furthermore, since it can be obtained from relatively inexpensive raw materials such as milk, mass production is possible.

  Lactoferrin in milk is industrially extracted from milk by separation techniques. The content of lactoferrin varies slightly depending on the production area, feed, etc., but it is usually a trace amount of 200 mg / kg in domestic milk. Therefore, it is not possible to procure enough milk in Japan to extract a small amount of lactoferrin, and it is not produced at all in Japan.

  The content of α-lactalbumin in milk is slightly different depending on the production area and feed, but is usually 1.2 g / kg for domestic milk. It is abundant in comparison with lactoferrin, and industrial separation is easy. As a result, stable securing is possible and procurement costs are low.

It is a figure which shows transition of the foot volume in a carrageenin foot edema suppression test model. The average value of 6 animals per group + standard error is shown. control represents control, WPI represents whey protein isolate, bLG represents β-lactoglobulin, aLA represents α-lactalbumin, and LF represents lactoferrin. *: Vs. control, p <0.05 (Dunnet's multiple comparison test) It is a figure which shows transition of the foot volume in an adjuvant arthritis (treatment) model. The average value of 6 animals per group + standard error is shown. Vehicle is a control, bLG is β-lactoglobulin, aLA is α-lactalbumin, LF is lactoferrin, and Dicro is diclofenac. *, **, ***: vs. Vehicle, p <0.05, 0.01, 0.001 (Dunnet's multiple comparison test) It is a figure which shows the frequency | count of a crying reaction in an adjuvant arthritis (pain) model. The average value of 6 animals per group + standard error is shown. Vehicle is a control, bLG is β-lactoglobulin, aLA is α-lactalbumin, 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 agony operation | movement in an acetic acid rising test model. The average value of 6 animals per group + standard error is shown. Vehicle is a control, WPI is a whey protein isolate, bLG is β-lactoglobulin, aLA is α-lactalbumin, 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 result regarding the pain level of the menstrual pain in the double blind crossover test which randomized the improvement effect with respect to the menstrual pain of (alpha) -lactalbumin. It is a figure which shows the result regarding the influence degree to the daily life by menstrual pain in the double blind crossover test which randomized the improvement effect with respect to menstrual pain of (alpha) -lactalbumin. It is a figure which shows the result regarding the use degree of an analgesic in the double blind crossover test which randomized the improvement effect with respect to menstrual pain of (alpha) -lactalbumin.

Hereinafter, the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely changed within the scope of the present invention.
The present invention relates to a COX-2 selective inhibitor comprising α-lactalbumin as an active ingredient. In the present invention, the COX-2 selective inhibitor means a substance having a COX-2 inhibitory effect stronger than the COX-1 inhibitory effect. The α-lactalbumin of the present invention is not particularly limited, but is preferably derived from human and / or non-human mammals (bovine, sheep, goat, horse, etc.), more preferably human or bovine α-lactoalcohol. It is albumin. The amino acid sequences and base sequences of these α-lactalbumins are already known and are registered in databases such as EMBL, DDBJ, NCBI. For example, bovine α-lactalbumin is registered with DDBJ as ACCESSION No. J05147. The base sequence of bovine α-lactalbumin is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. In the amino acid sequence shown in SEQ ID NO: 2, positions 1 to 19 are signal peptides. In addition, in the amino acid sequence shown in SEQ ID NO: 2, it is known that there is a mutant in which arginine (R) at position 29 is replaced with glutamine (Q) (“Milk General Dictionary” p.35, Asakura Shoten) ). Α-Lactalbumin secreted into milk is a mature peptide obtained by cleaving the signal peptide. Α-Lactalbumin used in Examples of the present application is the above-described bovine mature peptide.

  α-Lactalbumin is extracted from the milk of mammals such as humans, cows, sheep, and goats by ammonium sulfate precipitation ("Latest revised dairy technology manual", Dairy Technology Promotion Society, pages 122-125, 1975), ultrafiltration (patented) It can be produced by known techniques such as Document 3) and ion exchange method (Patent Document 4). The content of α-lactalbumin in milk varies slightly depending on the production area, feed, etc., but it is usually 1.2 g / kg for domestic milk, and if milk is used as a raw material for production, a large amount of α-lactalbumin is obtained. Can be prepared. Moreover, based on an amino acid sequence and a base sequence, you may synthesize | combine chemically or may manufacture by the genetic engineering method well-known to those skilled in the art. More simply, commercially available α-lactalbumin (for example, manufactured by Davisco) can be used.

Further, in the present invention, α-lactalbumin that can be used is not limited to the above-mentioned known amino acid sequence known as a natural form of a human or non-human mammal, as long as it has COX-2 selective inhibitory activity. It may be a mutant polypeptide having a mutation in the amino acid sequence. Examples of such polypeptides include natural mutants, homologs, artificial mutants, etc. Specifically, one or more amino acids are present in the natural α-lactalbumin amino acid sequence. List polypeptides that contain sequences that have additions, deletions, substitutions, or insertions, or that are encoded by polynucleotides that hybridize under stringent conditions to the complementary strand of a polynucleotide encoding native α-lactalbumin. More specifically, a polypeptide having a COX-2 selective inhibitory activity comprising a sequence in which one or more amino acids are added, deleted, substituted, or inserted into the amino acid sequence set forth in SEQ ID NO: 2. As another example, it is stringent to the complementary strand of a polynucleotide containing the coding region of the nucleotide sequence set forth in SEQ ID NO: 1. Hybridizing polynucleotide encodes at matter can include a polypeptide having a COX-2 selective inhibitory activity.
The mutant polypeptide can be produced by techniques well known to those skilled in the art. For example, total mRNA is prepared from animal cells that express α-lactalbumin, such as mammary gland, and RT-PCR is performed using primers designed based on the base sequence of natural α-lactalbumin. A mutant polypeptide can be obtained by introducing and expressing it in a host-vector system together with an appropriate promoter. Alternatively, a polynucleotide prepared by preparing a cDNA library from animal cells and using a probe prepared based on the base sequence of natural α-lactalbumin to hybridize to the probe from the cDNA library under stringent conditions And the polynucleotide is introduced into a suitable host and expressed, whereby a mutant polypeptide can be obtained. Those skilled in the art can appropriately select the promoter and host-vector system from known promoters and host-vector systems.
Those skilled in the art can appropriately select the stringent hybridization conditions. For example, in hybridization solution containing 25% formamide, 50% formamide under more severe conditions, 4x SSC, 50 mM Hepes pH 7.0, 10x Denhardt's solution, 20 μg / ml denatured salmon sperm DNA at 42 ° C. After performing overnight prehybridization, a labeled probe is added and hybridization is performed by incubating overnight at 42 ° C. The cleaning solution and temperature conditions in the subsequent cleaning are about “1 × SSC, 0.1% SDS, 37 ° C.”, and more stringent, highly stringent conditions are about “0.5 × SSC, 0.1% SDS, 42 ° C.” As severe stringent conditions, it can be carried out at about “0.2 × SSC, 0.1% SDS, 65 ° C.”. Thus, isolation of a polynucleotide having high homology with the probe sequence can be expected as the conditions for washing for hybridization become more severe. However, combinations of the above SSC, SDS, and temperature conditions are exemplary, and those skilled in the art will understand the above or other factors that determine the stringency of hybridization (eg, probe concentration, probe length, hybridization reaction). It is possible to achieve the same stringency as above by appropriately combining the time and the like.
A polypeptide encoded by a polynucleotide isolated using such a hybridization technique usually has a high homology in amino acid sequence with the natural polypeptide. High homology means homology of at least 40% or more, preferably 60% or more, more preferably 80% or more, more preferably 90% or more, more preferably at least 95%.
Alternatively, artificial mutations are introduced site-specifically or randomly into the base sequence of natural α-lactalbumin by cassette mutagenesis or PCR mutagenesis, and the polynucleotide is expressed using an appropriate host-vector system. By doing so, a mutant polypeptide 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. It can confirm by the method as described in an Example of this application. The amino acid sequence of such a mutant polypeptide having COX-2 selective inhibitory activity has high homology to the natural amino acid sequence, for example, the homology is 80% or more or 85% or more, Preferably it is 90% or more, More preferably, it is 95% or more.

  In this embodiment, as will be apparent from the examples described later, in order to enjoy anti-inflammatory analgesic action, α-lactalbumins, which are active ingredients, vary depending on the dosage form, symptoms, body weight, etc. 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 per kg of protein can be administered (taken).

  The dose of α-lactalbumin, which is an active ingredient of the anti-inflammatory analgesic of the present invention, is generally about 0.1 mg to about 50,000 mg per day as an active ingredient, preferably about 0.5 mg to about 10,000 mg, most preferably About 10 mg to about 2,000 mg can be administered at once or in portions to patients in need of treatment with the composition of the present invention before meals, after meals, between meals and / or before going to bed. it can. The dosage can also be determined individually depending on the age, weight and purpose of administration of the patient being administered.

The anti-inflammatory analgesic of the present invention can be used in the form of any medicine or food and drink. For example, it is expected to treat and / or prevent various inflammations and pains by directly administering as a pharmaceutical or by directly ingesting as a special-purpose food such as a food for specified health use or a nutritional functional food. In addition, various foods (milk, soft drinks, fermented milk, yoghurt, cheese, bread, biscuits, crackers, pizza crusts, prepared milk powder, liquid foods, for the sick, regardless of the form of liquid, paste, solid, powder, etc. Foods, nutritional foods, frozen foods, processed foods, and other commercially available foods).
The food containing the anti-inflammatory analgesic of the present invention can be used by mixing water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors and the like. Examples of proteins include whole milk powder, skim milk powder, partially skimmed milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin , Lactoferrin, soy protein, egg protein, meat protein and other animal and vegetable proteins, and their degradation products; various milk-derived components such as butter, whey minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipids, and lactose Etc. It may contain peptides and amino acids such as casein phosphopeptide, arginine and lysine. Examples of the saccharide include saccharides, processed starch (in addition to dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like. Examples of the lipid include animal oils such as lard, fish oil, etc., fractionated oils, hydrogenated oil, transesterified oil, etc .; palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, Examples include vegetable oils such as hydrogenated oils and transesterified oils. Examples of vitamins include 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. Examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, and selenium. Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid. In the production of foods and drinks containing the anti-inflammatory analgesic of the present invention, these may be either synthetic products or products derived from natural products, and / or foods containing a large amount may be used as raw materials. These components can be used in combination of two or more. The form of food may be solid or liquid. It may be in the form of a gel.
When the anti-inflammatory analgesic of the present invention is used as a pharmaceutical, it can be administered in various forms. Examples of the form include oral administration using tablets, capsules, granules, powders, syrups, etc., but they can be processed into preparations such as injections and liquids to be administered via tube, intravenous injection, intradermal, muscle Other dosage forms such as medium may be used. These various preparations are pharmaceutical preparations such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, solvents, isotonic agents, etc. It can be formulated using known adjuvants that can be commonly used in the technical field. Further, it may contain an appropriate amount of calcium. Further, an appropriate amount of vitamins, minerals, organic acids, sugars, amino acids, peptides, etc. may be added.

  The anti-inflammatory analgesic of the present invention can be used for the treatment of inflammatory diseases. The anti-inflammatory analgesic of the present invention can be applied to, for example, arthritis, rheumatoid arthritis (juvenile, chronic), ulcerative colitis, Crohn's disease, but is not limited thereto, and can be applied to all diseases involving inflammation. .

  There are various types of arthritis such as infectivity, immunity, and crystallinity. Specific examples include tuberculosis, gout, rheumatoid arthritis, osteoarthritis, acute rheumatic fever, psoriatic arthritis, juvenile rheumatoid arthritis, still disease, Reiter syndrome, ankylosing spondylitis, etc. , Goro Ogata, Fumiaki Takahisa, Seiichiro Tarui, “The Latest University of Internal Medicine Vol. 74 Joint Disease <Bone and Joint Disease 2>”, published by Nakayama Shoten, p.19, 1995). NSAIDs such as COX inhibitors are used as one of the agents used for the treatment of these diseases.

  Rheumatoid arthritis is a chronic inflammatory disease that causes multiple arthritis. The exact cause is unknown, but three predisposing factors, immunological factors, and environmental factors are regarded as important. In immunological factors, lymphocytes that secrete cytokines and play an important role in immunity function abnormally. As a result, abnormal antibodies appear and bind to the tissues of your joints, causing arthritis. It is supposed to cause. Many of the early symptoms are stiff joints when waking up, causing joint pain and swelling. There are many types of joint pain, including spontaneous pain (at rest), tenderness, and movement pain. Other symptoms include joint swelling, fever, and redness. Arthritis is synovitis in which the synovium is inflamed, and is often found symmetrically in the joints of the limbs. Commonly used joints are limbs, hands, feet, elbows, knees, shoulders, etc., and may deform as they progress. Once rheumatoid arthritis develops, complete treatment is difficult and patients will be associated with the disease for life.

  Today, drugs that are routinely prescribed to relieve inflammatory pain in joints such as rheumatoid arthritis or inflammatory pain at bone joint sites include aspirin called NSAIDs, indomethacin, ibuprofen, and diclofenac. Ten synthetic compounds and synthetic glucocorticoids called steroids.

  Prostaglandins mediate inflammatory pain, and inhibition of COX by NSAIDs to reduce / stop prostaglandin biosynthesis reduces pain. However, as already mentioned, these drugs frequently induce peptic ulcers when administered for a long time. Therefore, a method that can improve inflammatory pain in joints with a high degree of safety without inducing peptic ulcer has been sought for many years. A recent topic of NSAIDs is a COX-2 inhibitor that can specifically inhibit COX-2 induced at the site of inflammation and reduce peptic ulcers that were a side effect of NSAIDs (Hiroo Imura, Goro Ogata) , Shigeru Takahisa, Seiichiro Tarui, “The Latest University of Internal Medicine Vol. 74, Joint Disease <Bone and Joint Disease 2>”, published by Nakayama Shoten, p.19, 1995, and edited by Masanori Fukushima, “Merck Manual Medicine” Information home version ", Nikkei Business Publications, p.233-235, 1999).

  In addition, the anti-inflammatory analgesic of the present invention can be used effectively for alleviating pain without inflammation. There are two types of pain: acute pain and chronic pain. Acute pain is a warning signal that the body has been injured or damaged (such as an ankle sprain). It is a reaction that tries to remove the cause and prevent worsening by reacting to pain. Chronic pain, on the other hand, refers to the case where the warning signal does not play a role and the 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 involved in prostaglandin (PG) biosynthesis. There are currently at least three types of COX reported, COX-1, COX-2, and COX-3. COX-1 exists physiologically and controls physiological actions such as stomach and kidney. The concentration does not change regardless of stimuli such as LPS or inflammation. On the other hand, COX-2 is related to inflammation and tissue damage. Although it is not expressed in a normal state, it is newly produced mainly from migratory cells by stimulation or inflammation of LPS, IL-1, etc., and production is suppressed by dexamethasone. That is, when COX-2 is selectively inhibited, there are few side effects on the stomach, kidneys, and the like, and an effect of improving inflammation and tissue damage can be expected.

  Indications for COX inhibitors include (1) rheumatoid arthritis, osteoarthritis, fifty shoulders, cervical and shoulder syndrome, low back pain, tendonitis, gout, and other rheumatic diseases and motor organ diseases, and (2) postoperative / traumatic injury. Painful diseases such as after pain, cancer pain, dental pain, symptomatic neuralgia, stone pain, menstrual pain, (3) diseases with fever such as various infectious diseases, malignant tumors, collagen disease, (4 ) Indications using antithrombotic and antiplatelet effects such as cerebral infarction, transient ischemic attack, ischemic heart disease, Kawasaki disease, proteinuria, cancer metastasis, (5) endotoxin shock, patent ductus arteriosus , Low blood pressure, Bartter syndrome, male infertility, immunosuppression, indications mainly due to PG synthesis inhibitory action, such as strengthening of immunotherapy, etc. are known (Hiroo Imura, Goro Ogata, Fumiaki Takahisa, Kiichiro Tarui) , “Latest Internal Medicine Vol. 74, Joint Disease <Bone and Joint Disease 2>”, Medium Shoten, p.77-78, 1995 years). Therefore, the anti-inflammatory analgesic agent of the present invention having COX-2 inhibitory activity is applicable not only to all diseases accompanied by inflammation but also to the above various diseases.

  In addition, all prior art documents cited in the present specification are incorporated herein by reference.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited by this.

[Example 1] (Carrageenan paw edema suppression test, action on acute inflammation)
In this example, the inhibitory effects of whey protein isolate (WPI) and various proteins contained in WPI on carrageenan-induced foot edema were examined. WPI is a protein in which whey is processed by microfiltration (MF), ultrafiltration (UF), cross-follow microfiltration (CFM), ion exchange, etc., to separate proteins and increase the protein concentration.
(Method)
Various test substances were orally administered using Wistar rats (male, 6 weeks old, 1 group, 6 animals, 5 groups) that had been acclimated for 1 week or longer. As test substances, whey protein isolate dissolved in physiological saline (WPI, derived from bovine, manufactured by Davisco Foods International, Inc.) 1 g / kg [WPI group], α-lactalbumin (α-LA, derived from bovine, 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 (50 w / w% of WPI) [bLG group], lactoferrin (LF, derived from bovine, Murray Goulburn Ingredients) 10 mg / kg (1 w / w% of WPI) [LF group], and physiological saline [Control group] was administered at a dose of 4 ml / kg at the indicated dose. One hour after oral administration of the test substance, 0.1 mL of 1 w / w% carrageenan solution was intradermally administered to the left limb footpad. The foot volume was measured at 3 hours after carrageenin administration, and its anti-inflammatory effect was evaluated. In the following examples, WPI, α-LA, β-LG and LF were the same as those shown here.
(result)
The main components in WPI are α-lactalbumin (25 w / w% of WPI), β-lactoglobulin (50 w / w% of WPI), and lactoferrin (1 w / w% of WPI). This time, in order to examine the active ingredient in WPI, it was administered according to the content ratio. As a result, only α-lactalbumin was effective in suppressing carrageenan paw edema. From this result, it was found that α-lactalbumin contributes to the carrageenan foot edema inhibitory effect of WPI (FIG. 1).

[Example 2] (Adjuvant arthritis (treatment) model, effect on subacute inflammation (experimental chronic inflammation))
In this example, a rat adjuvant arthritis model, which is an animal model of rheumatoid arthritis, was created, and the therapeutic effects on arthritis of various proteins contained in WPI were verified.
(Method)
After the acclimation period of 1 week or longer, adjuvant arthritis was induced by subcutaneous injection of 0.1 mL of Freund's complete adjuvant into the left footpad of Wistar rats (male, 6 weeks old). The Freund's complete adjuvant used contains 8.5 mL of liquid paraffin, 80 1.5 mL of a surfactant span and 7.5 mg of human tuberculosis killed H37Rv in 10 mL. Fourteen days after administration of the adjuvant, rats with sufficient arthritis were selected and divided into groups based on the foot volume of the left limb footpad (1 group, 6 animals, 5 groups).
Test substances (α-lactalbumin [aLA group], β-lactoglobulin [bLG group], lactoferrin [LF group]) dissolved in physiological saline are each administered at a dose of 300 mg / kg, non-steroidal anti-inflammatory analgesics ( Diclofenac (manufactured by Wako Pure Chemical Industries, Ltd.) [positive control group] is an oral dose of 50 mg / kg for 3 consecutive days from adjuvant administration to the 14th to 16th day after administration of adjuvant in a volume of 4 ml / kg. Administered. As a control group, the same volume of physiological saline was orally administered for 3 consecutive days from the adjuvant administration to the 14th to 16th days.
On the 14th day (immediately after administration of the test substance), 15th and 17th days after administration of the adjuvant, the foot volume of the left footpad was measured to examine the therapeutic effect.
(result)
Α-lactalbumin suppressed the exacerbation of edema in contrast to edema of the left limb that received adjuvant. In addition, administration of lactoferrin decreased the foot volume, and a therapeutic effect was obtained (FIG. 2).

[Example 3] (Adjuvant arthritis (pain) model, action on chronic pain)
After the acclimation period of 1 week or longer, adjuvant arthritis was induced by subcutaneously injecting 0.1 mL of Freund's complete adjuvant similar to Example 2 into the left limb footpad of Wistar rats (male, 6 weeks old). Sixteen days after administration of the adjuvant, rats with sufficient arthritis were selected and divided into groups based on the foot volume of the left limb footpad (1 group, 6 animals, 5 groups).
Test substances (α-lactalbumin [aLA group], β-lactoglobulin [bLG group], lactoferrin [LF group]) dissolved in physiological saline are each 300 mg / kg, and the anti-inflammatory analgesic diclofenac [ The positive control group] was orally administered in a volume of 4 ml / kg at a dose of 50 mg / kg. One hour after administration of the test substance, the presence or absence of a crying response to bending extension stimulation pain in the left hind limb ankle was measured. The effect of pain suppression was evaluated by quantifying the number of times of crying when bent and extended 10 times.
(result)
An arthritic pain suppressing effect was obtained by single administration of α-lactalbumin and single administration of lactoferrin (FIG. 3).

[Example 4] (Acetic acid rising test, effect on pain)
In this example, the therapeutic effects of various proteins contained in WPI on acetic acid-induced pain were verified.
Whey protein isolate (WPI) [WPI group], α-lactalbumin dissolved in physiological saline using mice (male, 6 weeks of age, 1 group, 6 animals, 5 groups) after acclimation period of 1 week or more [ALA group], β-lactoglobulin [bLG group], lactoferrin [LF group] each at a dose of 300 mg / kg, anti-inflammatory analgesic diclofenac [positive control (Dicro) group] is 50 mg / kg The dose was administered orally in a volume of 4 ml / kg. In addition, the same volume of physiological saline was orally administered as a control (Vehicle) group. One hour after oral administration of the test substance, 0.2 mL of a 0.6 v / v% acetic acid solution was intraperitoneally administered. The pain suppression effect was evaluated by quantifying the number of 20-minute agony motions from 10 minutes to 30 minutes after acetic acid administration.
(result)
The results are shown in FIG. α-Lactalbumin and lactoferrin showed significant analgesic effects.

[Example 5] (Enzyme inhibition experiment)
In this example, the inhibitory activity of test substances (α-lactalbumin, WPI) on various enzymes related to inflammation was examined.
(Method)
(1) Activity measurement using an enzyme preparation of cyclooxygenase-1 (COX-1) (n = 2)
Various concentrations of test substance (α-lactalbumin or WPI) and cyclooxygenase-1 (EC 1.14.99.1, derived from human platelets) in 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 EIA (enzyme immunoassay). In addition, a test group (α-lactalbumin or WPI) not added was provided as a control group. The enzyme inhibition rate of α-lactalbumin and WPI was calculated with the average value of the measured values of the control group as 100% enzyme inhibition rate, and the 50% inhibition concentration was taken as the IC ₅₀ value.
(2) Activity measurement using an enzyme preparation of cyclooxygenase-2 (COX-2) (n = 2)
Various concentrations of test substance (α-lactalbumin or WPI) and cyclooxygenase-2 (EC 1.14.99.1, human recombination; Sf21 insect cells) enzyme preparation, 1 mM glutathione, 1 μM hematin, 500 μM phenol After preincubating in 100 mM Tris-HCl buffer (pH 7.7) for 15 minutes at 37 ° C., arachidonic acid was added to a final concentration of 0.3 μM and reacted at 37 ° C. for 5 minutes. After the reaction, the PGE ₂ concentration was measured by EIA. In addition, a test group (α-lactalbumin or WPI) not added was provided as a control group. The enzyme inhibition rate of α-lactalbumin and WPI was calculated with the average value of the measured values of the control group as 100% enzyme inhibition rate, and the 50% inhibition concentration was taken as the IC ₅₀ value.
(3) Activity measurement using enzyme preparation of inducible nitric oxide synthase (iNOS) (n = 2)
Various concentrations of test substances (α-lactalbumin or WPI) and inducible nitric oxide synthase (EC 1.14.13.39, derived from mouse macrophages) were prepared using 4 mM NADPH (nicotinamide adenine dinucleotide reduced form), 8 Preincubation for 15 minutes at 37 ° C in 100 mM Tris-HCl buffer (pH 7.9) containing µM (6R) -5,6,7,8-tetrahydro-L-biopterin, 8 µM FAD, 6 mM DTT (Dithiothreitol) Thereafter, 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 spectrophotometrically. In addition, a test group (α-lactalbumin or WPI) not added was provided as a control group. The enzyme inhibition rate of α-lactalbumin and WPI was calculated with the average value of the quantitative values of the control group as the enzyme inhibition rate of 100%, and the 50% inhibition concentration was taken as the IC ₅₀ value.
(4) Activity measurement using an enzyme preparation of phospholipase A2 (PLA2) (n = 2)
Various concentrations of test substances (α-lactalbumin 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 the rattlesnake) After pre-incubation in 0.1 M Glycine-NaOH buffer (pH 9.0) containing 20 μM EDTA at 37 ° C for 15 minutes, 1-palmitoyl-2- [1- ¹⁴ C] oleoyl-L-3-phosphatidylcholine The mixture was added to 0.03 μCi and reacted at 37 ° C. for 5 minutes. After removing unreacted 1-palmitoyl-2- [1- ¹⁴ C] oleoyl-L-3-phosphatidylcholine, the radioactivity of the oleate fraction was measured with a liquid scintillation counter. In addition, a test group (α-lactalbumin or WPI) not added was provided as a control group. The enzyme inhibition rate of α-lactalbumin and WPI was calculated with the average value of radioactivity (Ci) of the control group as the enzyme inhibition rate of 100%, and the 50% inhibition concentration was taken as the IC ₅₀ value.

(result)
The results are shown in Tables 1 and 2. α-Lactalbumin was highly active in cyclooxygenase-2, phospholipase A2-I, and phospholipase A2-II. In particular, cyclooxygenase-2 was found to be selectively highly active. In addition, α-lactalbumin was found to have high cyclooxygenase-2 activity even compared to WPI.

（表１ α-ラクトアルブミンの酵素阻害効果）

(Table 1 Enzyme inhibitory effect of α-lactalbumin)

（表２ WPIの酵素阻害効果）

(Table 2 Enzyme inhibitory effect of WPI)

[Example 6] (Human test for menstrual pain (pain))
In this example, the improvement effect of α-lactalbumin on menstrual pain was verified by a randomized double blind crossover test.
(subject)
A preliminary survey was conducted on 70 women who requested cooperation in posting of cooperation requests, and the menstrual cycle was stable for 28 days ± 3 days. A self-administered questionnaire about the latest menstruation (Table 3: Degree of pain of menstrual pain) , The degree of impact of daily menstrual pain on the daily life, use of analgesics) is the answer of menstrual pain 1 or higher, the menstrual pain score (degree of daily menstrual pain impact on the daily use of painkillers) 30 women (22-50 years old, average 36.4 years old) with a total of 2 or more were selected as subjects.
(Subject)
Tablets A and B with exactly the same appearance were prepared. One is α-lactalbumin tablet confectionery (containing 150 mg of α-lactalbumin in one tablet), and the other is one containing no α-lactalbumin (placebo tablet confectionery). Both the test subject and the evaluator started the test in a state in which which tablet confection was an α-lactalbumin tablet confection until the end of the evaluation.
(Intake method of the test object)
The test was carried out in the menstrual cycle from September to December 2007. The first menstrual cycle from the end of the first menstruation to the end of the next menstruation was established as the first test substance intake period. The menstrual cycle (from the end of menstruation to the end of the next menstruation) following the first subject ingestion period was a rest period in which no subject was ingested. Furthermore, the first menstrual cycle (from the end of menstruation to the end of the next menstruation) following the resting period was set as the second test substance intake period.
The subjects were divided into two groups, 1 group (tablet A intake period-rest period-tablet confection B intake period, n = 13) and 2 groups (tablet B intake period-rest period-tablet confection A intake period) N = 17), and the order of intake of tablet confection A and tablet confection B was set differently.
In the test substance intake period, two test articles (Tablet A or Tablets B) were continuously ingested three times in the morning, noon and evening.
The use of analgesics was allowed before the start of the test and during the test period.
(Evaluation method)
During one menstrual period before the start of the test, the subject was asked to complete a self-administered questionnaire (Table 3). In addition, subjects were continuously recorded on a self-administered questionnaire once a day during the test substance intake and rest periods. The content of the self-administered questionnaire is about menstrual pain and quality of life. The degree of menstrual pain entered in the self-administered questionnaire during the menstrual period, the impact on daily life, and the degree of use of analgesics are quantified using the verbal rating scale, and the maximum value during each period is evaluated. Used for. The menstrual pain to be evaluated was limited to back pain and lower abdominal pain.
Furthermore, the average value of the results of menstruation and the rest period before the start of the test was determined, and this was used as the value in the non-intake period.
(Statistical processing)
The average value and standard error were obtained for each measured value, and the significant difference was tested as a corresponding group by a non-parametric method using statistical software. (Wilcoxson signed rank test)
(result)
The verbal rating scale value, which measured the degree of menstrual pain, significantly decreased (p <0.01) when α-lactalbumin tablet confection was ingested compared to the non-ingestion period (FIG. 5).
The verbal rating scale value for evaluating the degree of influence (QOL) on the daily life due to menstrual pain tended to decrease (p <0.10) when α-lactalbumin tablet confection was consumed compared to the non-intake period (Fig. 6). ).
As for the degree of use of analgesics, 21 people (70%) took analgesics due to menstrual pain before the start of the study, but 10 in the intake period of α-lactalbumin tablets and placebo tablets. It decreased to people (33% of the total). The barval rating scale value, which measured the degree of use of analgesics, was significantly decreased in both tablet candy ingestion periods compared to the non-ingestion period (p <0.01) (FIG. 7).
Based on the above results, taking α-lactalbumin 300 mg three times a day (morning, noon, evening) significantly reduces the proportion of analgesics used, and alleviates menstrual pain and improves QOL Became.

（表３ 自記式質問紙の内容）

(Table 3 Contents of self-administered questionnaire)

  The α-lactalbumins which are active ingredients in the present invention can be mass-produced from relatively inexpensive raw materials such as milk. Moreover, since there are few side effects, it is safe and can be ingested over a long period of time, the anti-inflammatory analgesic effect can be enjoyed by mixing with a pharmaceutical or ingesting as a food. In addition, since sufficient nutritional management is possible by daily administration or ingestion, it can also be applied to uses such as the production of functional foods for patients with low nutritional status.

Claims (10)

A COX-2 selective inhibitor comprising α-lactalbumin as an active ingredient. An anti-inflammatory analgesic comprising α-lactalbumin as an active ingredient. Use of α-lactalbumin for the manufacture of anti-inflammatory analgesics. The anti-inflammatory analgesic according to claim 2, which is a therapeutic agent for inflammatory diseases. The anti-inflammatory analgesic according to claim 4, wherein the inflammatory disease is arthritis or rheumatoid arthritis. The anti-inflammatory analgesic according to claim 2, which is a therapeutic agent for painful diseases. The anti-inflammatory analgesic according to claim 6, wherein the painful disease is menstrual pain. An anti-inflammatory analgesic food or drink comprising α-lactalbumin as an active ingredient. A method for treating inflammatory diseases, wherein a therapeutically effective amount of α-lactalbumin is administered to a mammal. A method for treating painful diseases, comprising administering a therapeutically effective amount of α-lactalbumin to a mammal.

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