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    • 专利摘要:
    PURPOSE: A preventing and therapeutic agent for arthritis and joint injury containing glucose and amino acid as an effective ingredient and inducing collagen synthesis from chondrocyte cells by synergism thereof is provided which can effectively and economically treat arthritis(arthrosis) and various diseases related with joint injury. CONSTITUTION: This preventing and therapeutic agent contains 15 to 70% by volume of glucose having a concentration of 2.5 to 50%, 20 to 84.95% by volume of one or two or more of amino acids having a concentration of 2.5 to 50%, selected from proline, glycine, glutamine, alanine, aspartic acid, serine, methionine, arginine, lysine, cysteine and hydroxyproline, and 0.045 to 1% by weight of one or more of trace elements selected from the group consisting of Zn, Cu and Mn.
    公开号:KR20020072491A
    申请号:KR1020010069266
    申请日:2001-11-07
    公开日:2002-09-16
    发明作者:이일훈；임시웅
    申请人:이일훈；임시웅；
    IPC主号:
    专利说明:

    Prevention and treatment of arthritis and joint damage by synergistic action of glucose and amino acid {AN ARTHRITIS AND ARTHROSIS INJURY PREVENTION AND TREATMENT SOLUTION BY SYNERGISTIC ACTION OF GLUCOSE AND AMINO ACID}
    [4] The present invention relates to a prophylactic and therapeutic agent for arthritis and joint damage due to the synergistic effect of inducing collagen synthesis in chondrocytes using glucose and amino acids as active ingredients. More specifically, the present invention relates to a preventive and therapeutic agent for arthritis and joint damage, which can regenerate cartilage simply and efficiently by using amino acids and other substances in combination with high concentrations of glucose used to harden tendons or ligaments. will be.
    [5] Collagen is the most common protein constituting the structural support of the human or mammal's body, the basic component unit is tropocollagen protein. Tropocollagen consists of three polypeptide chains of the same size, each of which is wound around each other to form a superhelical cable or triple-twist helical rod. In tropocollagen, each of the three chains consists of about 1,000 amino acid residues.
    [6] Some various types of collagen proteins are commonly recognized as unique and varying amino acid compositions and lengths. Collagen type I consists of two α-1 (I) polypeptide chains and one α-2 polypeptide chain, and most of them are found in skin tissues, tendons, bone support structures and eye corneas.
    [7] Collagen type II consists of three polypeptide chains of type α-1 (II), which are found mainly in the cartilage of the joints, in the intervertebral discs and in the vitreous body of the eye. Collagen type III has three α-1 (III) polypeptide chains, one α-2 (IV) polypeptide chain and one α-2 (V) polypeptide chain, and is found in placenta and skin, for example. Other types of collagen have similar structural differences with each other.
    [8] On the other hand, according to a recent study, C-proteinase is a basic essential enzyme that catalyzes the cleavage of C-propeptide of fibrillar collagen, including collagen I, II and III. Here, 'C-proteinase' means a substance capable of treating collagen molecules, derivatives or fragments thereof, or precursors thereof by cleaving through -Ala ↓ Asp-Asp- and / or -Gly ↓ Asp-Glu-. do.
    [9] Arthritis is often divided into inflammatory or non-inflammatory arthritis. A representative disease of non-inflammatory arthritis is osteoarthritis (arthritis), and a representative disease of inflammatory arthritis is rheumatoid arthritis.
    [10] The osteoarthritis mainly occurs in people over 40 years old, and the etiology is known to be caused by the action of structural biomechanical factors in people with age, histological predisposition and genetic predisposition. Pathological findings include articular cartilage destruction accompanied by increased marginal osteophyte and subchondral bone, and more advanced osteoarthritis results in narrower joint cavity, resulting in irreversible structural deformation of the joint structure. do.
    [11] Patients with osteoarthritis have great restrictions on their daily activities and are unable to participate in leisure and sports activities.
    [12] Existing treatments for such osteoarthritis mainly focus on relieving symptoms and slowing the progression of the disease.Therapeutic drugs include anti-inflammatory and analgesics, recently developed cartilage protective agents (oral glucosamine, chondroitin sulfate and topical hyaluronic acid joint cavity). Injection).
    [13] In this case, when the anti-inflammatory agent is administered, there are gastrointestinal disorders (for example, gastritis, gastric ulcer, duodenal ulcer), hepatotoxicity, nephrotoxicity, hypertension, and edema, and when administering analgesics, side effects such as hepatotoxicity are concerned. In addition, hyaluronic acid in the cartilage protector is less effective in sustaining the effect, and also not satisfactory in terms of effect. Glucosamine and chondroitin have also not been fully tested in terms of effectiveness.
    [14] On the other hand, rheumatoid arthritis is a cell-mediated autoimmune disease, that is, a condition in which the immune system misdiagnoses the body's own tissue as an external tissue and initiates an abnormal immune response thereto. Rheumatoid arthritis is characterized by inflammatory synovitis, which causes cartilage destruction and fracture corrosion, resulting in permanent structural deformation.
    [15] Patients with rheumatoid arthritis may experience pain in joint swelling, inflammation, stiffness, and range of motion. In the advanced stage of arthritis, most of the range of motion may be lost, and life may be dependent on caregivers.
    [16] Common treatments for such rheumatoid arthritis include the use of non-specific cytotoxic immunosuppressants. This is because a large number of patients have CD4 + type T-cells, especially those that have a reaction with collagen and / or an abnormal humoral response to collagen. These drugs inhibit the entire immune system and selectively inhibit aberrant autoimmune responses. Makes things impossible.
    [17] Over time, the overall suppression of this immune system increases the risk for infection. Examples of such immunosuppressants include, but are not limited to, methotrexate, cyclophosphamide, Imuran (azathioprine) and cyclosporin A.
    [18] On the other hand, steroid compounds such as prednisone and methylprednisolone (also non-specific immunosuppressive and anti-inflammatory drugs) are also used for symptomatic relief. However, steroids also have a number of toxic side effects that are evident in long-term use.
    [19] In addition, prolo therapy, which has been recently performed in the United States, injects glucose into ligaments and injured ligaments to multiply ligaments and tendons and restores them. This has been found to improve. However, even if this method is applied to the tendon and ligaments, but the recovery of the fiber, but the articular cartilage regeneration is incomplete, so there are many limitations in the treatment of joint damage, such as arthritis.
    [20] Therefore, the conventional treatments used for joint damage such as arthritis have limited effectiveness, have obvious toxic side effects, and their effectiveness is limited because they cannot be used continuously for a long time, thus overcoming the disadvantages of existing treatments. There is an urgent need for treatment or treatment.
    [21] On the other hand, the pharmacological action of the known amino acids has been reported to be used as a medicine that supplements the decrease in the amino acid in the blood when the amino acid is administered in the body, and has the effect of improving the motor function, reducing fatigue and fatigue after exercise. (Korean Publication No. 99-77107), In fact, although it has been used in various medicines in Korea, it has not been reported that the amino acid itself has an excellent effect in the treatment of arthritis and joint damage.
    [22] The present inventors added glucose and amino acids, preferably vitamin C and other microelements, to induce the regeneration of articular cartilage, the main component of collagen, and to inhibit bone resorption to normalize the structure of the joint in arthritis. In addition to reducing the tissue inflammation and promote regeneration, the present invention was completed.
    [23] That is, an object of the present invention is to provide a new preventive and therapeutic agent for arthritis (arthrosis) and joint damage, which overcomes limitations such as one or more side effects and potential effects of the conventional arthritis (arthritis) treatment method.
    [24] Another object of the present invention is to provide a new prophylactic and therapeutic agent for arthritis (arthritis) and joint damage, which can induce collagen synthesis in articular chondrocytes and also reduce inflammation of synovial cells surrounding the joints.
    [25] It is yet another object of the present invention to provide new prophylactic and therapeutic agents for arthritis (arthritis) and joint damage that significantly reduce the clinical symptoms of arthritis, such as arthralgia and limited range of motion.
    [1] Figure 1 is an optical micrograph (200x magnification) showing the results of immunostaining for collagen type II, 1 (a) is a normal rabbit, 1 (b) is a photograph of the treatment group rabbit.
    [2] FIG. 2 is an optical micrograph (100 magnification) of the cartilage of the medial tibial plateau, where 2 (a) is a normal rabbit, 2 (b) is a treatment rabbit, and 2 (c) is a control rabbit. It is a photograph.
    [3] 3 is a scanning electron micrograph (4600 magnification) of the cartilage surface of the medial tibial plateau, where 3 (a) is a normal rabbit, 3 (b) is a treatment rabbit, and 3 (c) is a control rabbit.
    [26] According to one aspect of the present invention for achieving the above object,
    [27] By volume, from 10% to 90% glucose in the range of 2.5-50%; And
    [28] One or two or more amino acids 90 to 10% selected from proline, glycine, glutamine, alanine, aspartic acid, serine, glutamine, methionine, arginine, lysine, cysteine and hydroxyproline and having a concentration in the range of 2.5-50% It provides a preventive and therapeutic agent for arthritis and joint damage by synergistic action of glucose and amino acids, characterized in that consisting of.
    [29] According to the second aspect of the present invention,
    [30] 15-70% glucose by volume, in the range 2.5-50% by volume;
    [31] Vitamin C 0.05-10%; And
    [32] One or two or more amino acids 20 to 84.95% selected from proline, glycine, glutamine, alanine, aspartic acid, serine, glutamine, methionine, arginine, lysine, cysteine and hydroxyproline and having a concentration in the range of 2.5-50% It provides a preventive and therapeutic agent for arthritis and joint damage by synergistic action of glucose and amino acids, characterized in that consisting of.
    [33] According to the third aspect of the present invention,
    [34] On a volume basis, 20-60% glucose with a concentration in the range of 2.5-50%;
    [35] Vitamin C 0.05-10%;
    [36] 0.045 to 1% of at least one trace element selected from the group consisting of Zn, Cu and Mn; And
    [37] One or two or more amino acids 29 to 79.905% selected from proline, glycine, glutamine, alanine, aspartic acid, serine, glutamine, methionine, arginine, lysine, cysteine and hydroxyproline and having a concentration in the range of 2.5-50% It provides a preventive and therapeutic agent for arthritis and joint damage by synergistic action of glucose and amino acids, characterized in that consisting of.
    [38] First, the arthritis regeneration mechanism of the present invention will be described. The hypothesis can be divided into two parts.
    [39] First, Insulin-like growth factor (IGF-1), transforming growth factor (TGF-β), and basic fibroblast growth factor (b-FGF) are secreted by the composition mixed according to the present invention, and the cyclin D1 promoter (Cyclin) is sequentially D1 promoter) is expressed. This expression causes proliferation of chondrocytes and fibroblasts in the resting phase, and the fibroblasts are surrounded by the cartilage matrix secreted therefrom, and the fibroblasts then return to chondrocytes.
    [40] Second, b-FGF (basic Fibroblast Growth Factor) is secreted from chondrocytes and fibroblasts by the composition mixed by the present invention, and sequentially MEK (MAPK / ERK kinase, Mitogen-Activated Protein Kinase / Extracellular signal-Regulated Kinase) kinase and Mitogen-Activated Protein Kinase (MAPK) / ERK pathways are activated.
    [41] This results in the expression of a highly mobile group of DNA-binding predominant regions, which contain transcription factors expressed in all prechondrocytic and chondrocytes during Sox9 gene-embryonic development. . Expression begins in mesenchymal chondrogenitor cells. Then, collagen type II, VIII, VIII is synthesized in chondrocytes, resulting in the regeneration of cartilage.
    [42] Furthermore, the composition of the present invention selected in connection with the hypothesis mechanism comprises glucose; And amino acids; It consists of mixing certain vitamin C and other trace elements (Zn, Cu, Mn) in specific ratios.
    [43] Glucose, which is used as an active ingredient in the present invention, stimulates the transcription of TGF-β, ERK, and PKC (protein kinase C) through other metabolic pathways to produce extracellular matrix, the content of which is It is preferable that it is 2.5 to 50% of range.
    [44] If the concentration of glucose is less than 2.5%, the concentration is too low, so it is not sufficient to stimulate the transcription of growth factors such as TGF-β or fibroblast growth factor, and if the concentration exceeds 50%, The mucus is too thick and can cause pain in the patient and is not suitable for use as a drug. In addition, the volume of glucose used is determined according to the concentration of the liquid phase used, the volume in the concentration range is preferably 10 to 90%, more preferably 15 to 70%, most preferably 20 to 70% It is good to contain.
    [45] In addition, the amino acid used as a kind of the active ingredient in the present invention performs synergism of synthesizing collagen through gene expression together with cartilage growth factor secreted by glucose by using together with glucose, a kind of amino acid that can be used All 22 species are considered to be possible.
    [46] However, based on amino acid sequencing, it may be more desirable to selectively use amino acids involved in the growth factors required, such as proline, glycine, glutamine and alanine. (Ala), aspartic acid (Asp), serine (Ser), glutamate (Glu), methionine (Met), arginine (Arg), lysine (Lys), cysteine (Cys) and hydroxyproline (hydroxy-Pro) Among 12 kinds of amino acids, one kind or a combination of two or more kinds can be used as necessary.
    [47] For reference, the physiological and biochemical mechanisms of amino acids to date are not so many, but the amino acids are separated in relation to gene expression and nutrition supply of cells as follows.
    [48] . Maximum expression of IGF-1: 1) Dose dependence: arginine, proline
    [49] 2) Very low concentration: lysine
    [50] . ERK Activation, a Subclass of MAPK in Bovine and Human Articular Cartilage Cells: Cysteine
    [51] . Indirect Control of Collagen Gene Expression in Cultured Human Fibroblasts: Glutamine
    [52] . Chondrocytes Maturation Delay, Cartilage Mineralization and Osteoinhibition: Glutamate
    [53] . Synthetic Stimulation of Proteoglycans in Human Articular Cartilage Cells: Methionine
    [54] . Lysomal Pathway Inhibition: Glutamine, Methionine
    [55] . Substrates necessary for collagen synthesis: hydroxyproline, glycine, aspartic acid,
    [56] Alanine, Serine
    [57] The amino acids used in the present invention can affect gene expression while being itself a substrate of collagen synthesis.
    [58] In addition to the 12 amino acids described above, for example, asparagine (Asn), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), tryptophan (Trp), threonine (Thr), histidine (His) Other amino acids, such as Val and Val, can also be used if needed. It is preferable that the amino acid used for this invention is especially L-amino acid.
    [59] In addition, the 12 types of amino acids mentioned above may be used individually, and 2 or more types may be mixed and used for them. It is believed that when mixing two or more kinds, the volume percentage of the amino acid itself in the whole mixture composition can be properly determined according to the amino acid composition ratio and concentration of collagen type II by those skilled in the art.
    [60] The volume of the amino acid used in the present invention is in the range of 10 to 90% by volume, more preferably 20 to 84.95% by volume, and most preferably 29 to 79.905% by volume when the concentration range of the amino acid is 2.5 to 50%. good. The concentrations and thus volume ranges are selected ranges to prevent negative feed back, and adding less than this is not sufficient to express the gene and beyond this range is undesirable due to overall toxicity.
    [61] In addition, the concentration range of the amino acid in the volume range is more preferably 5 to 40% and most preferably within the range of 7.5 to 30%.
    [62] Furthermore, a summary of the metabolism and adverse effects revealed so far for some of the above-described amino acids are shown in Table 1 below.
    [63] TABLE 1
    [64] amino acidMetabolismAdverse effect Alanine (Ala)Urea, lactate, glucoseAmmonia Hyperemia Arginine (Arg)Urea, Ornithine, Citrulline, NO, PolyamineHypotension, tumor irritation, acidosis, hyperkalemia, cardiac arrest Aspartate (Asp)Urea, Asparagine, GlucoseNeurotoxicity Glutamate (Glu)Urea, Glutamine, GlucoseHypercholesterolemia, fatty liver, neurotoxicity GlycineSerine, NH4 +Neurotoxicity, Sodium Hyperemia LysineSaccharopin, amino adipateArginine Antagonism, Interstitial Nephritis Methionine (Met)Cysteine, homocysteine, cystationHomocysteine hyperemia, serum folate deficiency
    [65] In view of this adverse effect, the proportion of each amino acid in the 12 amino acid mixtures can be set as follows: proline 5 to 95%, glycine 10 to 90%, glutamine 1 to 50%, alanine 1 to 30%. , Aspartic acid 1-30%, serine 1-30%, glutamate 1-30%, methionine 1-50%, arginine 1-50%, lysine 1-50%, cysteine 1-50% and hydroxyproline 5-95 %. If it is added below this range, it is not enough to express the gene, and beyond this range, it is not desirable because of its overall toxicity.
    [66] Further preferred proportions of each amino acid are as follows: proline 10-80%, glycine 15-85%, glutamine 2-30%, alanine 2-20%, aspartic acid 2-20%, serine 2-20 %, Glutamate 1.5-15%, methionine 2-30%, arginine 2-20%, lysine 2-20%, cysteine 2-20% and hydroxyproline 7-80%.
    [67] Furthermore, the proportions of each of the most preferred amino acids are as follows: 15-70% proline, 17-60% glycine, 5-20% glutamine, 3-10% alanine, 3-10% aspartic acid, 3-10% serine %, Glutamate 2-10%, methionine 3-10%, arginine 3-10%, lysine 3-10%, cysteine 3-10% and hydroxyproline 9-70%.
    [68] When combining two or more types of amino acids, commercially available amino acids may be mixed at the predetermined ratios described above. Although the temperature at which the composition of the present invention is prepared and preserved is not particularly limited, it is preferable to prepare and store at room temperature or less.
    [69] In the present invention, vitamin C may be added to impart a protective effect on joint degeneration and to stimulate cell proliferation. At this time, the content is preferably 0.05 to 10% by volume, but less than 0.05% is not preferred as a coenzyme, if it exceeds 10% is not preferable because there is a risk of side effects such as hypervitaminemia.
    [70] On the other hand, the concentration of the vitamin C is preferably added in the range of 0.01 to 1000 mg / ml, preferably 0.1 to 500 mg / ml, and more preferably 1 to 300 mg / ml.
    [71] In addition, in the present invention, one or more selected from the group consisting of Zn, Cu and Mn as a trace element may be added in an amount of 0.045 to 1% based on volume.
    [72] Here, Zn causes division and differential growth to convert fibroblasts to chondrocytes, Cu assists the maturation of collagen, and Mn plays a major role in the synthesis of chondroitin sulfate, a major component of articular cartilage.
    [73] Each of these contents is, for example, in the range of 10 -10 to 1 mg / dL, more preferably in the range of 10 -8 to 0.7 mg / dL and most preferably 10 -7 to 0.3 mg / d, for Zn and Cu, respectively. within the range of dL, and in the case of Mn in the range of 10 -20 to 10 -1 mg / dL, more preferably in the range of 10 -15 to 10 -2 mg / dL and most preferably in the range of 10 -10 to It is recommended to add within the range of 10 -3 mg / dL. For reference, such trace elements are typically included in amino acids, etc., and the volume and content ranges are calculated when the range values are added separately.
    [74] The prophylactic and therapeutic agents for arthritis or joint damage produced by the present invention are useful as foods such as medicines and beverages. Although the dosage form in the case of using as a medicine is not specifically limited, It can go through general administration routes, such as oral administration, injection, and administration by fluid.
    [75] In the case of oral administration, it may be used as a composition having the above composition or as a preparation such as tablets, pills, capsules, gels, and syrups together with a pharmaceutically acceptable carrier and excipient. However, solid preparations such as tablets and powders often take a long time to be absorbed, and oral administration by liquid preparations is preferable. In that case, it is preferable to administer it as aqueous solution with a suitable additive, for example, salts, such as sodium chloride, a buffer, a chelating agent, etc.
    [76] Injectables may be added dropwise into the joint cavity or intravenously, for example, by adding appropriate buffers, isotonic agents, and the like dissolved in sterile distilled water.
    [77] The sugar core can be made by appropriate coating. For this purpose, concentrated sugar solutions can be used, optionally with gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. And the like. Pigments or dyes may be added to identify the active compound and to characterize different combinations.
    [78] Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin and soft capsules made of plasticizers such as gelatin and glycerol or sorbitol. Push-fit capsules may include the active ingredient and fillers such as lactose, binders such as starch, lubricants such as talc or magnesium stearate or optionally stabilizers. Soft capsules may be dissolved or suspended in an appropriate liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
    [79] The drug can also be administered by a target drug transport system such as liposomes coated with specific antibodies targeting arthritis or fibrosis tissue. Liposomes can be selectively taken in diseased tissue.
    [80] When used as a food, an appropriate flavor is added to encapsulate a powder prepared by a drink, for example, a soft drink or a powder drink, for example, a spray dry method, a freeze-drying method, a micropowder method, or the like. Can be.
    [81] Therapeutic effective amounts in the compositions of the present invention are first evaluated in cell culture assays. For example, it can be made in an animal model to obtain a calculated concentration range comprising IC 50 as determined in cell culture (eg the concentration of the test compound to obtain half of the maximum inhibition of activity). Such information can be used to determine more accurate dosages available to humans.
    [82] A therapeutically effective dose means an amount of a compound that reduces symptoms in a patient. The toxicity and therapeutic effects of these compounds are standard in cell culture or laboratory animals to determine LD 50 (a dose that can kill 50% of a population) and ED 50 (a dose that is effective for 50% of a population). It can be determined by a pharmaceutical process.
    [83] The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD 50 and ED 50 . Compounds with high therapeutic indices are effective. The data obtained from these cell culture assays and animal studies can be formulated in a range of dosages available for human use. Such dosages are appropriately within a range of mild concentrations that include non-toxic or mild ED 50 .
    [84] Dosages vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage are selected by the attending physician depending on the condition of the patient.
    [85] Meanwhile, the administration interval is determined using the MEC value. The therapeutic agents of the invention are administered using a method that maintains plasma levels above MEC for 10-90%, preferably 30-90%, most preferably 50-90% of the time. In the case of topical administration or screening acquisition, the effective local concentration of the drug is independent of the plasma concentration.
    [86] The amount of the administered composition can also vary depending on the individual being treated, the weight of the individual, the severity of the condition, the method of administration and the judgment of the physician. For example, the composition of the present invention can be administered at intervals of 1 week to 3 weeks at the site of arthritis of less than 10cc per time.
    [87] If necessary, the composition of the present invention may be provided in a package or administration device containing one or more unit dosage forms containing the active ingredient. Compositions composed of the compounds of the present invention formulated in a pharmaceutically acceptable carrier are suitably prepared and placed in suitable containers and indicated for the treatment of the indicated conditions. Appropriate indications indicated in the levels include treatment of arthritis and prevention of joint damage.
    [88] <Example>
    [89] The compositions of the present invention can be synthesized according to known techniques. Provided are methods for synthesizing and testing the compositions of the claimed invention.
    [90] Example 1-1. Composition synthesis
    [91] In the method for synthesizing the arthritis therapeutic composition according to the present invention, each amino acid was weighed in an amount necessary for the mixed composition using an electronic balance, dissolved in distilled water, and sterilized with high pressure steam. Then, glucose, vitamin C and trace elements were added to the amino acid solution dissolved in distilled water to prepare a therapeutic agent.
    [92] For example, after weighing in the amounts shown in Table 2, it was dissolved in 100 ml of distilled water and sterilized by high pressure steam.
    [93] TABLE 2
    [94] Amino acid compositionComponent content in 100 ml Proline glycine glutamine alanine aspartic acid serine glutamate methionine arginine lysine cysteine hydroxyproline4g4g2g1g1g1g0.5g1.5g1g1g1g2g Total content20 g
    [95] The desired therapeutic agent was prepared by adding glucose, vitamin C, and trace elements to the 20% concentration of the obtained amino acid solution in the amounts shown in Table 3 below.
    [96] TABLE 3
    [97] ingredientContent (%) of the ingredient in 100 mlVolume occupied by the corresponding ingredient within 0.5cc of total volume (% by volume) glucose20 g (20%)0.25 cc (50%) Vitamin c5 g (5%)0.05 cc (10%) Amino Acids Containing Trace Elements20 g (20%)0.20 cc (40%)
    [98] Example 1-2. Animal model for determination of prevention and treatment effect of arthritis.
    [99] In this example, a rabbit having chondrocytes most similar to human chondrocytes was used as a model. Specifically, 10 female New Zealand white rabbits (average weight: 3.4 ± 0.3kg, 8 ± 1 month old) were used. It was. The reason for using a mature rabbit was selected because it is similar to the condition causing osteoarthritis in humans and its cartilage regeneration ability is lower than that of immature rabbits.
    [100] The knees of these rabbits were left for 6 weeks after transverse cutting of the ipsilateral ACL. Of these, 5 rabbits did not perform separate knee arthritis treatment as a control group, and artificial saline was induced artificially with saline every 6, 8, 10, 13, and 16 weeks only to give stress equivalent to the treatment group. Injection into the knee joint.
    [101] In addition, the remaining five rabbits were treated knee arthritis artificially induced arthritis every 6 weeks, 8 weeks, 10 weeks, 13 weeks and 16 weeks as the therapeutic group of the present invention prepared in Example 1-1 after the induction of knee arthritis as a treatment group Injected into the joint.
    [102] Example 2. Measurement of Joint Regeneration
    [103] Prior to the experiment, the subjects focused on the medial tibial plateau, which is known as the site of most osteoarthritis.
    [104] (1) Biochemical Labeling Research
    [105] The medial tibial plateau of the control and treated rabbits of Example 1-2 at 19 weeks after the start of normal and treatment was evaluated by immunostaining to evaluate the degree of collagen type II synthesis. 1 is shown at 200 magnification. At this time, the rabbits of the control group had a severe degree of cartilage destruction and were not immunostained at all.
    [106] As shown in Figure 1, the therapeutic bacteria (1b) was confirmed that the cartilage was regenerated without a significant difference with the normal rabbit (1a).
    [107] (2) histopathological examination
    [108] The grades of the medial tibial plateau and lateral femur condyle of the control and treated rabbits of Example 1-2 were measured 19 weeks after the start of treatment using the histological grading system of Mankin et al. . The higher the grade, the more severe the destruction of articular cartilage.
    [109] TABLE 4
    [110] Rating I. Structural normal surface Slightly irregular surface Irregular surface Slightly irregular surface In gap region In gap radiation region In gap gap calcification region In gap transition region Loss In radiation region Loss calcification region Loss Complete dismantling012345678910 Cell 1. Contact area Normal cell swelling cell disappearance 2. Transitional and Radiation Regions Normal Cells Hypercellularity Cells Hypercellular Deep Cells Very High Cloning Cloning Deep Cloning Cells Hypocellularity Cell Deficient Cells Deep Cell Death012012345678910 Ⅲ.No common formation of pannus0123
    [111] On the other hand, the results of the examination of the tissue of the artificial joint with an optical microscope (100 magnification) based on the above table is shown in Table 5.
    [112] TABLE 5
    [113] ControlTreatment group Medial femoral condyle10.3 ± 0013.0 ± 001.7 ± 001.7 ± 00 Tibial plateau flat medial lateral15.3 ± 0011.3 ± 001.7 ± 001.7 ± 00
    [114] (However, the normal group is based on 0.0 ± 00 in both the femoral projections and the cervical plateau)
    [115] As shown in the above table, it was confirmed that cartilage regeneration is significantly improved in the composition treatment group.
    [116] Meanwhile, optical micrographs of artificial cartilage were shown in FIG. 2 according to histological examination of H & E. Here, 2 (a) is normal, 2 (b) is a treatment group, and 2 (c) is a control picture. Even when viewed with the naked eye, the control picture of 2 (c) shows that cartilage is completely destroyed in the weight-loading region.
    [117] In contrast, the treatment group (2 (b)) treated with the composition according to the present invention was confirmed that the cartilage was regenerated in the weight-bearing region and integrity was maintained. However, compared to normal cartilage, the arrangement of chondrocytes is somewhat reduced, but since cells have polarity, it is thought that the normal arrangement will recover over time.
    [118] (3) scanning electron micrograph
    [119] Scanning electron micrographs at 4600 times magnification were taken for normal, control and treated rabbits 19 weeks after the start of treatment and the results are shown in FIG. 3.
    [120] As clearly seen in Figure 3, compared to the normal (3a), the control group (3c) was confirmed that the cartilage is completely destroyed in the weight-bearing region is exposed to the subchondral bone (subchondral bone).
    [121] In comparison, the treatment group (3 (b)) treated with the composition according to the present invention was found to exhibit cartilage integrity above the normal level, although its integration was lower than normal in the weight-bearing region.
    [122] Example 3 Composition of Glucose, Vitamin C and Trace Elements
    [123] In Table 3, the same experiment as in Example 1-1 was repeated except that glucose, vitamin C, and trace element values were combined in an amount as described in Table 6 below (where amino acids are shown in Table 2). The composition described in the above was used as it is). In addition, for each of the therapeutic agents obtained, the experiment of the (1) biochemical labeling study of Example 2 was repeated and the results are shown together in the following Tables 6a to 6c.
    [124] TABLE 6a
    [125] Vitamin C content: 3% by volume Zn content: Amino acid content (% by volume) with a concentration of 0.1% by volume 2.5% = 100-vitamin C-Zn- below glucose content value Glucose at 10% concentration (% by volume)Biochemical Labeling Results 8%× 10%○ 35%○ 90%○ 95%×
    [126] (Circle): Excellent regeneration of cartilage, (triangle | delta): Normal regeneration of cartilage, x: Almost no regeneration of cartilage.
    [127] As shown in the table, it was confirmed that the content of glucose according to the present invention is preferably 10 to 90% by volume.
    [128] TABLE 6b
    [129] 35 vol% Mn content of 2.5% glucose: 0.1 vol% Amino acid content (% volume) with 10% concentration = 100-glucose content-Mn- vitamin C content Vitamin C (% by volume)Biochemical Labeling Results 0.03%× 0.05%○ 3%○ 10%○ 15%×
    [130] (Circle): Excellent regeneration of cartilage, (triangle | delta): Normal regeneration of cartilage, x: Almost no regeneration of cartilage.
    [131] As shown in the table, it was confirmed that the content of vitamin C according to the present invention is preferably 0.05 to 10% by volume.
    [132] TABLE 6c
    [133] 35% by volume glucose (vitamin C content) with 50% concentration: 3% by volume amino acid content (vol%) with 50% concentration = 100-glucose content-vitamin C content-below Cu content Cu (% by volume)Biochemical Labeling Results 0.003%× 0.045%○ 0.1%○ One%○ 3%×
    [134] (Circle): Excellent regeneration of cartilage, (triangle | delta): Normal regeneration of cartilage, x: Almost no regeneration of cartilage.
    [135] As shown in the table, it was confirmed that the content of the trace element according to the present invention is preferably 0.045 to 1% by volume. In addition, as the trace element, as can be seen from the above table, it may be selected and used from Zn, Mn, Cu as necessary.
    [136] Example 4 When No Trace Elements (Zn, Mn, Cu) were Added
    [137] First, the amino acid is 55% by volume while using the composition shown in Table 2, 10% by volume of vitamin C and 35% by volume of glucose with a concentration of 15% using the therapeutic agent of Example 2 ( 1) As a result of the biochemical marker research experiment, it was confirmed that the excellent cartilage regeneration ability. As a result, it was found that the trace element can be added as needed.
    [138] Example 5 No Vitamin C and Trace Elements (Zn, Mn, Cu) Added
    [139] Except for using vitamin C in Example 4, using 90% by volume of amino acid at 20% concentration and 10% by volume of glucose, the same method as described in Example 4 was repeated and then the same example Two (1) biochemical labeling experiments were performed.
    [140] At this time, the amino acid was used in 10% by volume and glucose was used in 90% by volume to combine another therapeutic agent, and the above experiment was repeated, and the (1) biochemical labeling experiment of Example 2 was performed.
    [141] As a result of the experiment, both cases were able to confirm excellent cartilage regeneration, and as a result it was confirmed that vitamin C can also be added as needed.
    [142] Therefore, as can be seen from the results of Examples 3 to 5 described above, it was confirmed that it is preferable to add glucose and amino acids in the concentration range of 2.5 to 50%, respectively, in the range of 10 to 90% by volume.
    [143] As a result, it was confirmed that the biochemical test, histopathology test and scanning electron microscopy were all excellent in cartilage regeneration when the therapeutic agent for joint damage of the present invention was used. In addition, in the above embodiment, but the experiments for arthritis, but is not limited to this, it will be understood that it will be effective in preventing and treating cartilage damage, such as meniscus damage, etc.
    [144] According to the above, by inducing the synthesis of synergistic collagen containing glucose and amino acids as an active ingredient, all diseases associated with arthritis (arthritis) and joint damage can be effectively and economically treated, and the prevention is also easy to forget.
    权利要求:
    Claims (6)
    [1" claim-type="Currently amended] By volume, from 10% to 90% glucose in the range of 2.5-50%; And
    One or two or more amino acids 90 to 10% selected from proline, glycine, glutamine, alanine, aspartic acid, serine, glutamine, methionine, arginine, lysine, cysteine and hydroxyproline and having a concentration in the range of 2.5-50% Prevention and treatment of arthritis and joint damage by synergistic action of glucose and amino acids, characterized in that consisting of
    [2" claim-type="Currently amended] 15-70% glucose by volume, in the range 2.5-50% by volume;
    Vitamin C 0.05-10%; And
    20% to 84.95% of one or more amino acids selected from proline, glycine, glutamine, alanine, aspartic acid, serine, glutamine, methionine, arginine, lysine, cysteine and hydroxyproline and having a concentration in the range of 2.5-50% Prevention and treatment of arthritis and joint damage by synergistic action of glucose and amino acids, characterized in that consisting of
    [3" claim-type="Currently amended] On a volume basis, 20-60% glucose with a concentration in the range of 2.5-50%;
    Vitamin C 0.05-10%;
    0.045 to 1% of at least one trace element selected from the group consisting of Zn, Cu and Mn; And
    One or two or more amino acids 29 to 79.905% selected from proline, glycine, glutamine, alanine, aspartic acid, serine, glutamine, methionine, arginine, lysine, cysteine and hydroxyproline and having a concentration in the range of 2.5-50% Prevention and treatment of arthritis and joint damage by synergistic action of glucose and amino acids, characterized in that consisting of
    [4" claim-type="Currently amended] According to any one of claims 1 to 3, when combining two or more kinds in the mixture of the amino acids, the proportion of each amino acid is 5 to 95% proline, 10 to 90% glycine, 1 to 50% glutamine, alanine 1-30%, 1-30% aspartic acid, 1-30% serine, 1-30% glutamate, 1-50% methionine, 1-50% arginine, 1-50% lysine, 1-50% cysteine and hydroxy Proline 5 to 95%, selected from among the prevention and treatment of arthritis and joint damage by synergistic action of glucose and amino acids
    [5" claim-type="Currently amended] According to any one of claims 1 to 3, wherein the concentration of vitamin C is 0.1mg / ㎖ ~ 1000mg / ㎖ characterized in that the prevention and treatment of arthritis and joint damage by synergistic action of glucose and amino acids
    [6" claim-type="Currently amended] The concentration of Zn and Cu in the trace element according to any one of claims 1 to 3 10 each-10˜1 mg / dL and the concentration of Mn is 10-20To 10-OnePrevention and treatment of arthritis and joint damage by synergistic action of glucose and amino acid, characterized in that mg / dL
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    同族专利:
    公开号 | 公开日
    KR100420560B1|2004-03-02|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2001-03-10|Priority to KR20010012405
    2001-03-10|Priority to KR1020010012405
    2001-11-07|Application filed by 이일훈, 임시웅
    2002-09-16|Publication of KR20020072491A
    2004-03-02|Application granted
    2004-03-02|Publication of KR100420560B1
    优先权:
    申请号 | 申请日 | 专利标题
    KR20010012405|2001-03-10|
    KR1020010012405|2001-03-10|
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