KR20080069732A - Orotate and orotate derivatives of enhancing biosynthesis of hyaluronic acid and/or glycosaminoglycan - Google Patents

Abstract

A composition comprising orotic acid or a derivative thereof is provided to enhance biosynthesis of hyaluronic acid and/or glycosaminoglycan in the cartilage cells, synovial cells, myofibroblasts, etc, and to prevent or treat arthritis, prolapsed intervertebral disk, winkles, dry eye syndrome, atopic dermatitis, etc. A composition for enhancing biosynthesis of hyaluronic acid and/or glycosaminoglycan comprises at least one selected from orotic acid and derivatives thereof as an active ingredient. The orotic acid derivative is at least one selected from the group consisting of orotates of sodium, potassium, calcium, magnesium, zinc, manganese, selenium, chrome, lithium, cobalt, iron and ammonium, choline orotate, creatine orotate, carnitine orotate, arginine orotate, lysine orotate, chitosan orotate, chitosan oligo orotate, polylysine orotate, orotidylate(orotidine monophosphate), orotidine and dihydroorotic acid.

Description

Orotic and orotate derivatives of enhancing biosynthesis of hyaluronic acid and / or glycosaminoglycan} to promote biosynthesis of hyaluronic acid and / or glycosaminoglycan

1 is a graph illustrating the degree of hyaluronic acid synthesis in chondrocytes, synovial membrane cells and fibroblasts induced by orotic acid.

Figure 2 is a graph comparing the degree of recovery of goblet cells after treatment with choline orotate in the dry eye animal model compared with the positive control group.

3 is a tissue staining comparison of the recovery of goblet cells after treatment with choline orotate in the dry eye animal model.

The present invention includes arthritis, discs (intervertebral discs), skin aging and the like comprising an active ingredient of orotic and orrotic acid derivatives that induce the synthesis of hyaluronic acid and / or glycosaminoglycans in animals and human cells, animals and the human body. It relates to the prevention and treatment of wrinkles, dry eye and ophthalmic diseases, wound healing, scar removal, vaginal dryness, gum disease, interstitial cystitis, atopic dermatitis and dry skin.

The present invention is an arthritis, disc (intervertebral disc protrusion), skin aging and wrinkles, dry eye and eye disease, wound healing, scar removal, vaginal dryness, gum disease, interstitial cystitis, The present invention relates to a composition for preventing and treating atopic dermatitis and dry skin. More specifically, the present invention relates to atopic dermatitis and dry skin, and more particularly, to a composition for promoting biosynthesis of hyaluronic acid and / or glycosaminoglycan in animals and human cells, animals, and human bodies. Arthritis, disc (intervertebral disc protrusion), skin aging and wrinkles, dry eye and eye disease, wound healing, scar removal, vaginal dryness, gums containing orotic acid and orotic acid derivatives as active ingredients A prophylactic and therapeutic composition for diseases, interstitial cystitis, atopic dermatitis and dry skin.

Herein, glycosaminoglycan refers to polysaccharides including amino sugars, including chondroitin sulfate, keratan sulfate, heparin, heparan sulfate, and dermatan, including hyaluronic acid. Sulfuric acid (dermatan sulfate) and the like.

Hyaluronic acid and glycosaminoglycans are biopolymer polysaccharides present in all living organisms, and the human body is distributed in 56% of skin, 35% of muscle tissue, and 9% of joint synovial fluid and spinal fluid. As a substance, 1 molecule of hyaluronic acid polysaccharide combines with 218 water molecules to have both physical viscosity and elasticity, and performs various functions such as shock absorption and lubrication in muscles and joints of the skin. In addition, hyaluronic acid is an important body polysaccharide that binds to receptors of various cells such as CD44, RHAMM, and regulates life phenomena through cell differentiation, proliferation and cell signaling.

In our bodies, hyaluronic acid and glycosaminoglycans are broken down in the process of removing harmful oxygen caused by ultraviolet rays or inflammation every day, and more than 3g per day is maintained by a fast metabolism process, which is maintained again, but with age Gradually, the amount produced decreases. As the amount produced is less than the amount to be degraded, the skin loses its elasticity, wrinkles begin to form, and in the joints, shock absorption and lubrication are not performed properly, and the causes of degenerative diseases that gradually destroy cartilage and wear are continuously worsened. A vicious cycle occurs.

Until now, the development of medicines using hyaluronic acid and glycosaminoglycan has been mainly used as a surgical aid for maintaining eye shape during cataract surgery, anti-adhesion agent after surgery, anti-wrinkle injection, wound healing agent, sustained-release agent, and cell therapy agent, eyeball Dry eye and eye diseases Eye drops, interstitial cystitis, gum disease. In tissue engineering, various products are used safely as scaffords and cosmetic skin moisturizers by utilizing the physicochemical properties of hyaluronic acid and glycosaminoglycans. Recently, hyaluronic acid is used as a main ingredient in the field of health functional food and is used for skin moisturization and prevention of hyaluronic acid-related diseases.

Hyaluronic acid and glycosaminoglycans are synthesized through the biosynthesis of mucopolysaccharides in the biosynthetic pathway of amino sugars. Hyaluronic acid and glycosaminoglycan are synthesized through a series of metabolic processes using glucose or glycogen as a raw material. One molecule of UTP is used in the synthetic route, and it is known to play an important role in the biosynthesis of UTP sugar derivatives.

The pharmacological action of orotic acids known to date is useful for the deficiency of enzymes or for the lack of pyrimidine nucleotides and nucleic acid synthesis by the conversion of orotic acid to 5'-phosphate by the catalytic action of orotic acid kinase enzyme. Has been reported to be used as a medicament to reduce ortic aciduria (MATINDALE, 1989, 1627).

The inventors found that orotic acid and orotic acid derivatives are used in chondrocyte cells, fibroblast cells, synovial cells, epithelial and keratocytes of the eye, and / or glycosa. The present invention was completed by first confirming the efficacy of promoting biosynthesis of minoglycans.

The inventors completed the present invention for the first time to confirm that orotic acid and orotic acid derivatives are effective in promoting biosynthesis of hyaluronic acid and / or glycosaminoglycans in chondrocytes, synovial cells, fibroblasts, and corneal cells. Accordingly, an object of the present invention is to provide arthritis, discs (intervertebral discs), skin aging and the like, which include active ingredients of orotic and orrotic acid derivatives that induce hyaluronic acid and glycosaminoglycan synthesis in animals and human cells, animals and humans. It relates to the prevention and treatment of wrinkles, dry eye and ophthalmic diseases, wound healing, scar removal, vaginal dryness, gum disease, interstitial cystitis, atopic dermatitis and dry skin.

The present invention comprises arthritis, disc (intervertebral disc protruding disease), skin aging and wrinkles, dry eye, and containing orotic acid and orotic acid derivatives represented by the following formula (1) and a pharmaceutically acceptable carrier Ophthalmic diseases, wound healing, scar removal, vaginal dryness, gum disease, interstitial cystitis, atopic dermatitis and dry skin prevention and treatment compositions are characterized by the composition.

The present invention will be described in more detail as follows.

The present invention is effective for the prevention and treatment of arthritis, disc (intervertebral disc prolapse), skin aging and wrinkles, dry eye and eye disease, wound healing, scar removal, vaginal dryness, gum disease, interstitial cystitis, atopic dermatitis and dry skin Arthritis, disc (intervertebral disc protruding), skin aging and wrinkles, dry eye and ophthalmic diseases, which contain orotic acid and orotic acid derivatives as active ingredients, especially those that exhibit the synthetic effect of hyaluronic acid and / or glycosaminoglycans in cells It relates to a composition for preventing and treating wounds, wound healing, scar removal, vaginal dryness, gum disease, interstitial cystitis, atopic dermatitis and dry skin.

Orotic and orotic acid derivatives promote the production of hyaluronic acid and / or glycosaminoglycans in cells, leading to arthritis, discs (intervertebral disc protrusion), skin aging and wrinkles, dry eye and ophthalmic diseases, wound healing, scar removal, vagina It will treat dryness, gum disease, interstitial cystitis, atopic dermatitis and dry skin.

Orotic and orrotic acid derivatives used in the present invention can be extracted from nature or prepared using synthetic methods.

R1 may independently form a hydrogen, a cation metal salt, a basic amino acid, a nontoxic water-soluble cation having a molecular weight of 1,000 Daltons or less, or an alkyl, alkene or alkoxy group having 1 to 20 carbon atoms.

Orotic acid and derivatives of orotic acid may include the following.

Specific examples of orotic salts of orotic acids (sodium, potassium, calcium, magnesium, zinc, manganese, selenium, chromium, lithium, copper, cobalt, iron, ammonium), choline orotate, creatine orotate, carnitine orotate, arginine orotate, methylglucamine These include orotate, ornithine orotate, lysine orotate, chitosan orotate, chitosan oligo orotate, polylysine orotate, orotidylate (orotidine monophosphate), orotidine, and dihydroorotic acid.

Thus, in the present invention, the in vivo synthesis of hyaluronic acid and / or glycosaminoglycans in chondrocytes, fibroblasts, cornea cells and synovial membrane cells for the physiologically imparted orotic and orotic acid derivatives were measured. It was confirmed that the compound showed excellent activity.

The present invention is easy to absorb into the cell based on the fact that the cellular self-synthesis capacity can be restored for a certain period of time through the injection of external hyaluronic acid, the degradation of the production capacity of hyaluronic acid and glycosaminoglycans of the cells generated during the aging process It relates to a new therapeutic substance that promotes biosynthetic mechanisms through orotic and orotic acid derivatives.

Here, existing studies on hyaluronic acid and glycosaminoglycan biosynthesis mechanisms include rate-determining steps in the intracellular synthesis process, and the activity of the enzymes involved in this biosynthesis reaction step determines the amount of biosynthesis of hyaluronic acid. Promoting substances increase the amount of hyaluronic acid and glycosaminoglycans in the body artificially and in a short time, resulting in decreased production and function of hyaluronic acid and glycosaminoglycans. ), Skin aging and wrinkles, dry eye and ophthalmic diseases, wound healing, scar removal, vaginal dryness, gum disease, interstitial cystitis, atopic dermatitis and dry skin treatments.

Here, arthritis refers to degenerative joints, osteoarthritis and rheumatoid arthritis due to aging, and refers to inflammatory diseases of joints due to lack of production of hyaluronic acid and glycosaminoglycans or small molecules in the joint cavity.

The efficacy of hyaluronic acid in arthritis has been clinically proven to be effective in relieving pain, eliminating inflammation, preventing cartilage tissue destruction, and lubricating, and recently, according to reports, the effect of cartilage regeneration on the proliferation of cartilage cells and the synthesis of cartilage matrix after long-term administration. Molecularly, the effects of hyaluronic acid injection have been found to inhibit inflammation and pain, to inhibit gene expression and activity of chondrogenic enzymes, and to differentiate and proliferate chondrocytes and increase biosynthesis of matrix substances.

Here, gum disease includes periodontal disease, which is a generic term for periodontitis, gingivitis, and gum wounds. Hyaluronic acid reported that hyaluronic acid is effective in treating periodontal and gum diseases. (J. clin. Periodontal. 2003, 30 (2): 159-64)

Dry eye and eye disease refer to diseases caused by lack of tears, hormonal abnormalities or aging, and diseases such as LASIK, cataracts and glaucoma surgery, and diseases such as conjunctivitis and keratitis.

Interstitial cystitis is a disease that causes urination disorders and pain due to wounds and inflammation in the epithelial cells inside the bladder, and it is reported that hyaluronic acid and glycosaminoglycans are applied to the wound site and have a significant healing effect. It was. (scan. J. urol. Nephrol. 2005, 39 (2), 143-147)

However, the externally injected hyaluronic acid does not directly exert its half-life in the body for about 12 hours, but stimulates Type B synovial cells of the synovial membrane of the joint synovial membrane and increases the biosynthesis of hyaluronic acid through intra-articular injection five times a week. Increases the amount of hyaluronic acid biosynthesis in the human body through the phenomenon that the amount of is changed to a healthy joint state, which indicates a therapeutic effect, and the production of hyaluronic acid gradually decreases within one year after treatment, and the symptoms of arthritis recur again. Has been found to be directly linked to disease treatment.

Diseases caused by decreased production and function of hyaluronic acid and glycosaminoglycans are inevitable for long-term administration. Fundamental solution is the development of effective drugs that can be taken in the long term without side effects. When hyaluronic acid is used directly, it has a limited effect that can only be seen as a local effect, but orotic acid and orotic acid derivatives have various routes of administration, so that the arthritis caused by the increase of hyaluronic acid and glycosaminoglycans in the whole body is a fast effect and safe treatment method. It can be developed for various uses such as disc (intervertebral disc protrusion), skin aging and wrinkles, dry eye and ophthalmic diseases, wound healing, scar removal, vaginal dryness, gum disease, interstitial cystitis, atopic dermatitis and dry skin prevention and treatment. have.

Therefore, arthritis, disc (intervertebral disc protruding syndrome), skin aging and wrinkles, dry eye and ophthalmic diseases, wound healing, scar removal, vaginal dryness, gums, containing orotic and orotic acid derivatives represented by Formula 1 as active ingredients Diseases, interstitial cystitis, atopic dermatitis and dry skin preventive and therapeutic compositions include arthritis, disc (intervertebral disc protrusion), skin aging and wrinkles, dry eye and ocular disease, wounds through activation of the rate-determining step of the amino acid synthesis process. Healing, scar removal, vaginal dryness, gum disease, interstitial cystitis, atopic dermatitis and dry skin can be prevented and treated, which makes it possible to develop various drugs for various diseases related to hyaluronic acid and glycosaminoglycan. It is supposed to be. The disease prevention and treatment composition related to hyaluronic acid and glycosaminoglycan by orotic acid and orotic acid derivatives according to the present invention contains the orotic and orrotic acid derivatives represented by Formula 1 as an active ingredient, As needed, along with acceptable carriers, arthritis, disc (intervertebral disc protrusion), skin aging and wrinkles, dry eye and ophthalmic diseases, wound healing, scar removal, vaginal dryness, gum disease, interstitial cystitis, atopic dermatitis and dry skin It can be prepared as a prophylactic and therapeutic agent.

Formulation Method of Orotic Acid and Orotic Acid Derivatives

The present invention also provides a composition of orotic and orotic acid derivatives comprising a pharmaceutically acceptable carrier, diluent, or excipient, or a combination thereof, which enhances the bioavailability of orotic and orrotic acid derivatives.

The term "pharmaceutical composition" means a mixture of the compound with other chemical components such as diluents or carriers. The pharmaceutical composition facilitates the administration and delivery of the compound into and out of the organism. There are a variety of techniques for administering and delivering compounds, including but not limited to oral, injection, aerosol, topical, mucosal and parenteral administration, and the like. Pharmaceutical compositions may also be obtained by reacting acid compounds such as hydrochloric acid, bromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

The term “therapeutically effective amount” is used to reduce or reduce to some extent one or more symptoms of the disorder being treated, or to delay the onset of a clinical marker or symptom of a disease that requires prevention. Means the amount of active ingredient. Thus, a pharmacologically effective amount may be to some extent reduce (1) the effect of reversing the rate of progression of the disease, (2) the effect of inhibiting further progression of the disease, and / or (3) one or more symptoms associated with the disease. It means the quantity which has an effect to remove (preferably). A pharmacologically effective amount can be determined empirically by testing the compound in known in vivo and in vitro model systems for diseases in need of treatment.

The term "carrier" is defined as a compound that facilitates the addition of a compound into a cell or tissue. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the incorporation of many organic compounds into cells or tissues of an organism.

The term "diluent" is defined as a compound that not only stabilizes the biologically active form of the compound of interest, but also is diluted in water to dissolve the compound. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline, because it mimics the salt state of human solutions. Because buffer salts can control the pH of a solution at low concentrations, buffer diluents rarely modify the biological activity of a compound.

The compounds used herein may be administered to a human patient as such or as a pharmaceutical composition mixed with other active ingredients as in combination therapy or with a suitable carrier or excipient. Techniques for formulation and administration can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, 18th edition, 1990.

Accordingly, the pharmaceutical compositions according to the invention employ one or more pharmaceutically acceptable carriers which comprise excipients or auxiliaries which facilitate the treatment of the active compounds into formulations which can be used pharmaceutically. It may be prepared by a conventional method. Proper formulation is dependent upon the route of administration chosen. Any of the known techniques, carriers and excipients can be used suitably and as understood in the art, for example in Remingston's Pharmaceutical Sciences described above. In the present invention, the compound of Formula 1 may be formulated into an injectable preparation, an oral preparation, and the like as desired.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, pathological condition, food, time of administration, route of administration, rate of excretion and responsiveness of the patient. Can be. The pharmaceutical composition of the above-mentioned prophylactic and therapeutic agents related to hyaluronic acid and glycosaminoglycan of the present invention contains the orotic and orrotic acid derivatives as active ingredients. The orotic and orrotic acid derivatives can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical formulations. That is, the orotic and orrotic acid derivatives of the present invention can be administered in various oral and parenteral formulations during actual clinical administration, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants and surfactants. It is prepared using diluents or excipients. Solid form preparations for oral administration include tablets, pills, powders, granules and capsules, and the like, which form at least one excipient such as lactose, sucrose, mannitol, or sorbitol in orotic and orrotic acid derivatives. Fillers such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragakenes, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethyl cellulose, and / or polyvinylpi Cellulose-based materials such as rolidone (PVP). If desired, carriers such as disintergrating agents such as crosslinked polyvinyl pyrrolidone, urticaria, or salts thereof such as alginic acid or sodium alginate and lubricants such as magnesium stearate, binders and the like may be added. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories as terms, including injections, ointments, patches and the like. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol and gelatin may be used.

Dosage units may contain, for example, one, two, three or four times the individual dosage, or they may contain 1/2, 1/3 or 1/4 times. The individual dosage preferably contains an amount in which the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose. Effective doses of orotic acid and orotic acid derivatives are concentration dependent but are preferably 0.1 mg to 1 g / kg, more preferably 0.4 to 200 mg / kg, and may be administered 1-6 times a day.

In addition, the present invention is an arthritis, disc (intervertebral disc protrusion), skin aging and wrinkles, dry eye and ophthalmic diseases, wound healing, scar removal, vaginal dryness, gum disease, interstitial containing orotic acid and orotic acid derivatives as an active ingredient It provides a health food composition for preventing and improving cystitis, atopic dermatitis and dry skin. In the present specification, the health food is a food which improves the function of the general food by adding orotic acid and orotic acid derivatives to the general food, and adding the orotic and orrotic acid derivatives to the general food, or encapsulating, powdering, suspension, etc. can do. When ingested, it brings a specific effect on health, and unlike the general medicine because it is made of food as a raw material has the advantage that there is no side effect that can occur when taking long-term use of the drug.

When the orotic and orrotic acid derivatives of the present invention are used as food additives, the orotic and orrotic acid derivatives may be added as they are or used together with other food or food ingredients, and may be appropriately used according to conventional methods. The mixed amount of the active ingredient can be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, orotic acid and orotic acid derivatives may be added in the amount of 0.0001 to 10% by weight, preferably 0.1 to 5% by weight based on the raw materials in the manufacture of food or beverage. However, in the case of prolonged ingestion for health and hygiene purposes or for health control purposes, the amount can be adjusted below the above range. In addition, when the health food of the present invention is used as the pharmaceutical composition, it is preferable to contain orotic acid and orotic acid derivatives within the measured toxicity range.

There is no particular limitation on the kind of food. Examples of foods to which the orotic acid and orotic acid derivatives can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, including ice cream, various soups, drinks , Teas, drinks, alcoholic beverages and vitamin complexes. Specifically, health foods containing orotic acid and orotic acid derivatives include health foods and favorite products such as juices, teas, jelly, juices, etc. which are mainly composed of orotic acid and orotic acid derivatives.

When the orotic acid and orotic acid derivative of the present invention is used as a cosmetic raw material, the orotic and orrotic acid derivatives may be added as they are or used together with other cosmetic ingredients, and may be appropriately used according to a conventional method. It can be suitably determined according to the purpose. In general, it can be added in an amount of 0.0001 to 10% by weight, preferably 0.1 to 5% by weight based on the raw materials in the manufacture of cosmetics using orotic and orrotic acid derivatives. Cosmetics can be applied to skins, lotions, creams, packs and cosmetics.

When the orotic and orotic acid derivatives of the present invention are used as raw materials for quasi-drugs such as toothpaste and gargle, the orotic and orrotic acid derivatives may be added as they are or used together with other ingredients, and may be appropriately used according to conventional methods. The amount of the mixture may be appropriately determined depending on the purpose of use thereof. Generally, the amount of 0.0001 to 10% by weight, preferably 0.1 to 5% by weight, based on the raw materials in preparation of toothpaste and goggles using orotic and orrotic derivatives. Can be added. As a quasi drug, it can be applied to toothpaste, mouthwash, and gargle.

Hereinafter, the present invention will be described in more detail based on the following examples, but the scope of the present invention is not limited only to the following examples.

Example  One : Orotic  Hyaluronic Acid Synthesis in Chondrocytes

1. Chondrocyte Separation

Cartilage tissues were isolated from aseptically isolated rabbits from 3 to 4 weeks old and treated with 0.2% collagenase type II at 37 ° C for 4 hours and then filtered with 100um filter to obtain cartilage cells. Cells were replaced every 2 days with chondrocytes (DMEM / F12, 10% FBS, Insulin, Nonessential amino acid, Ascorbic acid, penicillin, Gibco BRL products) at 37 ° C and 5% CO ₂ .

2. Make bead

Chondrocytes were mixed with 1.5% alginate solution to 2X10 ⁶ cell / ml, and then dropped into each drop of 102mM CaCl ₂ solution (pH7.4) using a syringe to make chondrocyte-bead, and finally washed with 0.15M NaCl. . Beads were replaced every 2 days before the experiment by adding cartilage culture medium (DMEM / F12, Nonessential amino acid, Ascorbic acid, Penicillin, Gibco BRL products) and orotic acid at 37 ℃ and 5% CO2.

3. Content of Hyaluronic Acid in Bead

3-1. Bead acquisition

Beads were cultured for 2 weeks by adding chondrocyte culture medium and orotic acid and washed with PBS. The beads were collected and treated in 55mM sodium citrate, 30mM EDTA, 150mM sodium chloride (pH6.8) solution at 4 ℃ for 10-15min, followed by Papain 20ug / ml in 0.1M sodium acetate, 50mM sodium EDTA, 5mM L-cyctein hydrochloride (pH5.53) was made to react overnight at 56-60 degreeC. For enzyme inhibition, the sample obtained after heating for 10 minutes was stored at -20 ℃ until the assay.

3-2. HA assay

One day prior to assay, HA-binding protein (HABP) was coated in a 96 well plate at 4 ° C overnight and blocked with 1% BSA. After binding the samples for 60 minutes at 37 ℃ washed with 0.5% PBST. After biotin-HABP was bound for 60 minutes at RT, extra-avidin alkaline phosphatase was added and then developed with p-nitrophenyl-phosphate (PNPP). This reaction is inhibited with 3N-NaOH and the absorbance is read at 405nm wavelength. See FIG. 1 for the results.

Example  2 : Orotic synovial  Hyaluronic Acid Production in Cells

Human synovial cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium containing 10% FBS (fetal bovine serum), and subcultured by adding 2 × 10 ⁴ cells / well to a 24well-plate. The next day, the cells were replaced with DMEM medium containing 5% FBS and incubated at 37 ° C. for 3 days with the addition of orotic acid. After 3 days of culture, the total hyaluronic acid content in the medium was measured by the sandwich ELIZA method of HA binding protein (HABP) and HA binding protein-biotin and read at 405 nm. Hyaluronic acid (SIGMA H) was used as a standard. See FIG. 1 for the results.

Example  3: Orotic  Hyaluronic Acid Synthesis in Fibroblasts

Rabbit fibroblasts were cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium containing 10% FBS (fetal bovine serum), and subcultured by adding 2 × 10 ⁴ cells / well to a ²⁴ well plate. The next day, orotic acid was added to DMEM medium containing 5% FBS and incubated at 37 ° C. for 3 days. After 3 days of culture, the total amount of hyaluronic acid in the medium was measured by sandwich ELIZA method of HA-binding protein (HABP) and HA-binding protein-biotin. Hyaluronic acid (SIGMA H) was used as a standard. See FIG. 1 for the results.

Example  4: eye Corneal cell  And corneal epithelial cell isolation

Clean up the tissue by removing blood vessels of the corneal tissue. After washing with 5% Antibiotics-PBS, the cleaned tissue was added to 3 ml of 1X Trypsin (0.05%) / EDTA (0.01%) and shaken incubated at 37 ° C. for 80 minutes. After collecting the suspension, SHEM media (10% FBS in DMEM / F12 (1: 1) + 0.5% PS + 4mM L-glutamine + 10ng / ml EGF + 5ug / ml insulin + 30ng / ml choleratoxin + 0.18mM adenine + 2nM 3.3 After washing with '.5 Triiodo-L-thyromine Sodium salt + 0.4ug / ml hydrocortisone), centrifuge at 1,200rpm for 5 minutes. The separated cells were used as epithelium and incubated with 1.2U / ml Dispase II at 37 ° C for 2 hours. After removing 1.2U / ml Dispase II, 5U of 200U / ml type I collagenase was treated, shaking incubation at 37 ° C. Collect the suspension and wash it with culture media (10% FBS in DMEM / F12 (1: 1) + 1% PS), centrifuge at 1,200rpm for 5 minutes and plate it in the dish.

Example 5 . In corneal parenchymal cells choline orotate Effect on the production of pseudo hyaluronic acid

Treated keratocytes with choline orotate (0uM ~ 10uM) for 48 hours, and then 100 μl of cell culture medium and 100ul of b-HABP (50 ng / ml) at room temperature. React time.

The day before the experiment, HA 20ug / ml, 20mM Na2CO3 solution was added to 96well plate, refrigerated for 24hr, washed 3 times with standard assay buffer (SAB), 1% BSA was added to 96well plate and reacted for 90 minutes at room temperature. Wash three times to prepare an HA coated plate.

The prepared cell culture solution is added to the coated plate and reacted with stirring at room temperature for 1 hour. After washing 3 times with SAB, Extra-avidin alkaline phosphatase (1: 80000) was added and reacted at room temperature for 30 minutes, and then washed 3 times with SAB. Add 1mg / ml disodium p-nitrophenyl solution dissolved in 100mM NaCl and 5mM MgCl solution and react for 20 ~ 60 minutes. The reaction was stopped by adding 3N NaOH and measuring at 405 nm. As shown in the figure, choline orotate-treated ocular corneal cells were found to promote the production of hyaluronic acid in a concentration-dependent manner.

choline orotate conc. (uM) Hyaluronic acid conc. (Ng / ml) 0 3.848 ± 0.82 One 5.97 ± 1.07 2.5 7.56 ± 0.69 5 9.23 ± 0.54 10 10.4 ± 1.26

Example 6 . choline orotate On by Glycosaminoglycans  ( glycosamino - glycan Impact on the generation of

After treatment with choline orotate (0uM ~ 50uM) to the keratocytes (48uM) for 48 hours, the cells were cultured and the glycosaminoglycans were measured at 550nm using a Biocolor assay kit. As shown in the table below, eye corneal cells treated with choline orortate were found to promote the production of glycosaminoglycans in a concentration-dependent manner.

choline orotate conc. (uM) Glycosaminoglycan conc. (Ng / ml) 0 3.78 ± 0.47 One 4.53 ± 0.39 2.5 6.56 ± 0.64 5 9.85 ± 0.50 10 10.36 ± 0.84

Example 7 . In corneal epithelial cells choline orotate Effect on the production of pseudo hyaluronic acid

Treat epithelial cells with choline orotate (0uM ~ 10uM) for 48 hours, and then take 100ul of cell culture medium and 100ul of b-HABP (biotin-hyaluonic acid binding protein, 50ng / ml) at room temperature. React time.

The day before the experiment, HA 20ug / ml, 20mM Na2CO3 solution was added to 96well plate, refrigerated for 24hr, washed 3 times with standard assay buffer (SAB), 1% BSA was added to 96well plate and reacted for 90 minutes at room temperature. Wash three times to prepare an HA coated plate.

The prepared cell culture solution is added to the coated plate and reacted with stirring at room temperature for 1 hour. After washing 3 times with SAB, Extra-avidin alkaline phosphatase (1: 80000) was added and reacted at room temperature for 30 minutes, and then washed 3 times with SAB. Add 1mg / ml disodium p-nitrophenyl solution dissolved in 100mM NaCl and 5mM MgCl solution and react for 20 ~ 60 minutes. The reaction was stopped by adding 3N NaOH and measuring at 405 nm. As shown in the table, ocular corneal epithelial cells treated with choline orotate were found to promote the production of hyaluronic acid in a concentration-dependent manner.

choline orotate conc. (uM) Hyaluronic acid conc. (Ng / ml) 0 0.89 ± 0.048 One 1.25 ± 0.057 2.5 1.63 ± 0.041 5 2.85 ± 0.069 10 2.68 ± 0.038

Example 8 . In epithelial cells choline orotate On by Glycosaminoglycans  Impact on generation

The epithelial cells of the eye were treated with choline orotate (0uM ~ 50uM) for 48 hours, and then cell cultures were taken and measured for glycosaminoglycans at 550 nm using a Biocolor assay kit. As shown in the table below, epithelial cells treated with choline orotate were found to promote the production of glycosaminoglycans in a concentration-dependent manner.

choline orotate conc. (uM) Glycosaminoglycan conc. (Ng / ml) 0 1.46 ± 0.32 One 4.84 ± 0.73 2.5 5.62 ± 0.42 5 6.85 ± 0.79 10 7.36 ± 0.92

Example 9 . in vivo  Animal testing

Concanavalin A was injected into the rabbit lacrimal gland to induce inflammation, causing dry eye and eye disease. (Journal of ocular pharmacology & therapeutics 21 (2), 139-148, 2005) 5% choline orotate was dissolved in physiological saline buffered with phosphate solution and used as eye drops. As a positive control, a commercially available 0.1% hyaluronic acid agent was used, and the control group was a physiological saline buffered with phosphate solution. Impression cytology was evaluated by measuring impression cytology using topical saline buffered with choline orotate, hyaluronic acid, and phosphate solutions in the eyes of rabbits with dry eye and eye disease. As shown in FIG. 2, it was confirmed that epithelial cells were significantly restored in impression cytology in the case of eye drops using choline orotate compared to the positive control group and the control group. As shown in FIG. 3, restoration of impression cytology cells before and after administration can be confirmed as a result of impression cytology tissue staining.

The present invention is an arthritis, disc (intervertebral disc protrusion), skin aging and wrinkles, dry eye using an active ingredient of orotic and orrotic acid derivatives to induce the promotion of hyaluronic acid and glycosaminoglycan synthesis in animals and human cells, animals and humans And ophthalmic diseases, wound healing, scar removal, vaginal dryness, gum disease, interstitial cystitis, atopic dermatitis and dry skin.

Claims (11)

Hyaluronic acid and / or glycosaminoglycan biosynthesis promoting composition comprising at least one selected from orotic or orotic derivatives as an active ingredient According to claim 1, wherein the orotic acid derivative is orotic salt (sodium, potassium, calcium, magnesium, zinc, manganese, selenium, chromium, lithium, copper, cobalt, iron, ammonium), choline orotate, creatine orotate, carnitine orotate, at least one selected from the group consisting of arginine orotate, lysine orotate, chitosan orotate, chitosan oligo orotate, polylysine orotate, orotidylate (orotidine monophosphate), orotidine, dihydroorotic acid According to claim 1, wherein the composition is arthritis, disc (intervertebral disc prolapse), skin aging and wrinkles, dry eye and eye diseases, wound healing, scar removal, vaginal dryness, gum disease, interstitial cystitis, atopic dermatitis And a preventive and therapeutic effect of dry skin. The composition of claim 1, wherein the composition comprises one or more pharmaceutically acceptable carriers or excipients. The composition according to claim 4, wherein the content of the active ingredient is administered in the range of 0.1 to 6,000 mg / day per 1 kg of adult body weight. For the treatment of arthritis, disc (intervertebral disc protrusion), skin aging and wrinkles, dry eye and ophthalmic diseases, wound healing, scar removal, vaginal dryness, gum disease, interstitial cystitis, atopic dermatitis and dry skin comprising the composition of claim 4 Formulation 7. The formulation according to claim 6, wherein the formulation comprises at least one pharmaceutically acceptable carrier or excipient. 8. The carrier according to claim 7, wherein the preparation is a filler corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragakenth, methyl cellulose, hydroxy such as lactose, sucrose, mannitol, or sorbitol. Cellulose-based materials such as propylmethyl-cellulose, sodium carboxymethyl cellulose, and / or polyvinylpyrrolidone (PVP), cross-linked polyvinyl pyrrolidone, urticaria, or alginic acid or sodium alginate Disintergrating agents such as salts thereof, lubricants such as magnesium stearate, solid carriers such as binders, distilled water, physiological saline, aqueous glucose solutions, alcohols such as ethanol, liquid carriers such as propylene glycol, and polyethylene glycol, and various animals and plants At least one carrier or excipient selected from the group consisting of oily carriers such as oil, white petrolatum, paraffin and wax Formulation, characterized in that The preparation according to claim 6 or 8, wherein the preparation is a powder, granule, solution, pill, troche, suspension, emulsion, syrup tablet, powder, hard or soft capsule, suspension, injectable preparation. , Emulsions, formulations for parenteral administration The arthritis according to claim 1, wherein the composition further comprises a pharmaceutically acceptable excipient in the form of a beverage, beverage additive or food, food additive or cosmetic, cosmetic additive or quasi-drug. Disc (intervertebral disc protrusion), dry eye and ophthalmic disease, cosmetic aid for wrinkle removal, prevention of adhesion after surgery, cosmetic moisturizing, artificial skin, wound healing, scar removal, vaginal dryness, cataract surgery aid, atopic dermatitis improvement or treatment composition The composition of claim 10, wherein the content of the active ingredient is included in a range of 0.0001 to 10% by weight.

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