TWI660735B - Use of a peptide having 4 or 5 amino acids for treating the degenerative joint disease - Google Patents

Abstract

The present invention refers to the use of a four/five peptide GEKG (F) for the treatment of degenerative arthritis.

Description

Use of four/five peptide GEKG(F) for the treatment of degenerative arthritis

The present invention discloses a novel use of the four/five peptide GEKG (F) for the treatment of degenerative arthritis.

Degenerative joint disease refers to a persistent lesion of articular cartilage accompanied by hyperplasia of the epiphyseal bone, most of which occurs under the edge of the joint or under the cartilage. The cause of degenerative arthritis is quite complex, but it is mainly related to the age of the patient at the time of onset. Degenerative arthritis often occurs in older elders. It was confirmed that the occurrence of degenerative arthritis was based on the detection of articular cartilage wear and fibrosis, occurrence of twin bones (spurs) and/or inflammation of soft tissues. Although it is known that age changes do affect cartilage lesions, the true pathogenesis of degenerative arthritis has not yet been clarified.

Most of the degenerative arthritis occurs in joints or interphalangeal joints that carry body weight, especially the terminal phalanx, where joint pain and activity limitation are clinically major signs. Pathological examination often reveals joint deformation and swelling of the joint edges and pain of contact and even joint sound.

Current drugs for the treatment of degenerative arthritis include non-steroidal anti-inflammatory agents (such as COX2 pharmaceutical preparations), intra-articular injection of steroids, and intra-articular injection of nutritional lubricants (ie, hyaluronic acid preparations).

No. 4,528,133 discloses the use of certain four peptides comprising 2 or 3 residues of alanine (Ala; A) for the treatment of arthritis.

US 6034057 discloses the use of certain cyclopeptides comprising 8 amino acid residues and 2 linking groups for the treatment of rheumatoid arthritis.

US 6589750 B2 and US Pat. No. 7,429,448 B2 disclose the use of a five-peptide QHNPR for preventing or treating water-mineral imbalances in tissues or organs, such as bone, wherein Q represents branine (Gln); H represents histidine ( His); N represents aspartic acid (Asn); P represents proline (Pro); and R represents arginine (Arg).

None of the prior art teaches or suggests that a four/five peptide rich in glycine (Gly; G) can be used to treat degenerative arthritis.

Therefore, the inventors have in-depth discussions on existing problems, and through years of research and development and manufacturing experience in related industries, through continuous efforts to improve and test, finally succeeded in developing a four-fifth peptide GEKG (F) for treatment degradation. The use of arthritis can provide a new option for the treatment of degenerative arthritis.

Accordingly, a primary object of the present invention is to provide a four/five peptide GEKG (F) for use in the treatment of degenerative arthritis.

Based on this, the present invention mainly achieves the foregoing objects and effects through the following technical means.

The following is a description of the preferred embodiments of the present invention, and the detailed description of the present invention will be described in detail with reference to the accompanying drawings. .

Figure 1 shows the effect of tetrapeptide on the DNA content of chondrocytes.

Figure 2 shows the effect of tetrapeptide on collagen synthesis in cultured cartilage tissue.

Figure 3 shows the effect of tetrapeptide on the synthesis of proteoglycan in cultured cartilage tissue.

The present invention is a use of a four/five peptide GEKG (F) for the treatment of degenerative arthritis. The technical contents of the present invention will be described below by way of specific embodiments, and those skilled in the art can readily understand the advantages and effects of the present invention from the disclosure of the present disclosure. However, the invention may be embodied or applied by other different embodiments. Variations in the design and needs of the specific embodiments of the invention may be made without departing from the scope of the invention as claimed.

The invention also discloses a pharmaceutical composition comprising a therapeutically effective amount of a four/five peptide GEKG (F) and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes solid carriers, semi-solid carriers and liquid carriers, of which the types and suitable amounts of such solid carriers, semi-solid carriers and liquid carriers are known in the art. And used. The pharmaceutical composition can be in the form of a tablet, tablet, pill, powder or granule. The pharmaceutical composition may also be in the form of a gel, paste or patch. The pharmaceutical composition may also be in the form of a water soluble buffer, including but not limited to an aqueous solution of phosphate or citrate.

Further, the present invention uses abbreviations as defined in the biochemical sciences: lysine (Lys; K); glutamic acid (Glu; E); and, phenylalanine (Phe; F).

The four peptides GEKG and the five peptides GEKGF disclosed in the present invention can be prepared according to the methods described in the prior art, such as TW 201129368 A1 and TW 201333045 A1.

The method for measuring SOD, NO and MDA in joint fluid used in the invention Methods and methods for measuring collagen and proteoglycan in chondrocyte stroma are well known and conventional in the art.

Implementation

Example 1

First, the animal model

Fifteen healthy adult New Zealand white rabbits were randomly divided into 3 groups: normal control group (group A), model control group (group B) and peptide injection group (group C), with 5 rats in each group. Fasting for 4 hours before surgery, intramuscular injection of Ketamine (10 mg/kg) and Atropine (0.2 mg/kg). After the anesthesia is effective, the animal is fixed on the operating table, 8cm longitudinal incision is made at the posterior edge of the patellofemoral ligament and the skin is lifted. The joint capsule and synovium are opened along the posterior edge of the kneecap, and the knee joint is bent by 90° to expose the lower end of the femur. Two cylindrical defects of 3 mm in diameter and 3 mm in depth were made, and the coagulation at the defect was removed. The joint cavity was cleaned with sterile physiological saline, and the layers were carefully sutured. Penicillin was injected for one week after surgery. After 5 days, you can eat normally and stand up on your own.

Second, the experimental method

At the 2nd week after surgery, group A and group B did not do any treatment. Group C was injected with 250 mg/kg intra-articular injection of four peptides, GEKG, every 7 days for 12 weeks. 0.5 ml of physiological saline was injected into the joint cavity, and 0.2 ml of joint fluid was collected. Cut the sac of the switch, take out the synovial membrane and completely remove the articular surface of the humerus, observe the degree of articular cartilage lesions under the naked eye and operative lens, and slice the hematoxylin-eosin (HEStain, Hematoxylin and Eosin Stain) and toluidine blue. Observed by an optical microscope.

Third, the experimental results

Visual observation

After 12 weeks, the knee joint of group A was smooth and elastic, and a few joint fluids were visible in the joint cavity. The defect group of group B was only partially repaired, but there were still depressions and gaps in the center of the defect; all defects in group C were completely repaired, and the section structure It is no different from normal joints. The surface cartilage and its underlying cancellous bone have been completely repaired, and no signs of ossification and repair have been seen.

Tissue morphology observation

The joint tissue of group A has the same characteristics as the normal joint tissue. The surface is mature cartilage tissue, the middle is a mature mature tarsal plate and the ossified cancellous bone is below, and the thickness of cartilage tissue is normal. The defect group of group B is The repaired tissue was mainly fibrillar cartilage and fibrous tissue, and the interface between the normal tissue and the normal tissue was continuous. Some specimens had more fibrous cartilage tissue filling in the defect area, but there was no obvious tarsal formation in the defect area and the cancellous bone below. Poor dysfunction; the repaired tissue of group C has no significant difference with the characteristics of normal joint tissue. The surface cartilage tissue, tarsal plate and lower cancellous bone have reached normal histological morphology. The thickness of new cartilage is the same as normal, and the junction of cartilage and bone has been Cannot distinguish.

Biochemical indicator test

Compared with group B, the morphology of articular cartilage in group C was significantly improved and SOD in joint fluid was significantly increased, while NO and MDA were significantly decreased.

in conclusion

1. Histomorphology experiments showed that the injection of the four peptides GEKG The damaged cartilage tissue can be obviously repaired in the joint cavity, and the treatment effect on degenerative arthritis is very accurate.

2, the four peptides GEKG significantly reduce the MDA in the synovial membrane, reduce the degree of lipid peroxidation damage in the joint cavity, thus reducing the damage of free radicals on chondrocytes and matrix, to protect the articular cartilage.

3, four peptides GEKG significantly increase the SOD concentration in the rabbit joint cavity, eliminate the free radicals produced by inflammation, and thus protect the cartilage and inhibit and prevent cartilage lesions.

4, the four peptides GEKG significantly increased the NO concentration in the rabbit joint cavity, thus reducing the destruction of cartilage by NO.

Example 2

First, the experimental method

1. The rabbit growth plate chondrocytes were taken for continuous culture, and the four peptides, GEKG alone, collagen alone, and the four peptides, GEKG+collagen, were added to add the four peptides, GEKG (250ppm) and collagen (100ng/). Ml) and four peptides of GEKG (250ppm) + collagen (100ng / ml), and set a negative control group, after the new cartilage tissue is over the bottom of the centrifuge tube, transfer to a 24-well culture plate to continue the culture, the next day, change the medium, After the scheduled incubation time (3, 7 and 14 days) is reached, the specimens are collected for each test.

2, histology and histochemical detection: collection of specimens for HE staining, immunohistochemistry (S-P method) detection.

3. Determine the DNA content of chondrocytes using the Hoechst 33258 method.

4, chondrocyte matrix synthesis: in addition to water, the most important component of the chondrocyte matrix is collagen and more protein composed of glycosaminoglycans and core proteins sugar. Matrix content and metabolic status were quantified by measuring the content of collagen and proteoglycan in new tissue.

5. Determination of collagen synthesis in chondrocytes by hydroxyproline method: Since the proportion of hydroxyproline in collagen is relatively constant (about 10% of the weight of collagen), the production of chondrocyte collagen can be analyzed by quantifying hydroxyproline. . Operate in strict accordance with the instructions of the hydroxyproline detection kit and calculate the collagen content of each sample.

Second, the experimental results

1. The effect of four peptides GEKG on chondrocyte proliferation: The addition of four peptides GEKG significantly increased the proliferation of chondrocytes, and the DNA content of chondrocytes showed statistical difference with the control group (Fig. 1). The results indicated that the four peptides GEKG could be used. Effectively promote chondrocyte proliferation.

2, the effect of four peptides GEKG on the synthesis of chondrocyte matrix: the addition of four peptides GEKG significantly promoted the synthesis of collagen and proteoglycans and further effectively promoted chondrocyte proliferation, the results were statistically different from the control group (Figure 2 and 3).

Therefore, it can be understood that the present invention is an innovative invention, which not only effectively solves the existing problems, but also greatly enhances the therapeutic efficacy, and does not see the same or similar product creation or public use in the same technical field, and has the effect at the same time. enhance. Therefore, the present invention has met the requirements for "novelty" and "progressiveness" of the invention patent, and is the invention of the invention patent.

<110> Fubby Biotech Co., Ltd.

<120> Use of four/five peptides GEKG(F) for the treatment of degenerative arthritis

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<170> PatentIn version 3.5

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<213> Artificial sequence

<223> Synthetic peptide

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Claims (3)

A use of a tetrapeptide, GEKG, for the preparation of a medicament for the treatment of degenerative arthritis, wherein G represents glycine (Gly), E represents glutamic acid (Glu) and K represents lysine (Lys), wherein the treatment is The SOD content in the joint fluid increased, the NO and MDA levels decreased, and the articular chondrocytes proliferated. The use of claim 1, wherein the drug is a pharmaceutical composition, wherein the pharmaceutical composition comprises a therapeutically effective amount of a tetrapeptide, GEKG, and a pharmaceutically acceptable carrier. The use of claim 2, wherein the pharmaceutical composition is a patch.