JP2018115138A - Novel use for treating osteoarthritis with tetrapeptide-3 gekg or pentapeptide-3 gekgf - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel therapeutic agent useful for the treatment of osteoarthritis.SOLUTION: A pharmaceutical composition contains tetrapeptide-3 GEKG (Gly-Glu-Lys-Gly) or pentapeptide-3 GEKGF (Gly-Glu-Lys-Gly-Phe) having a specific sequence as an active ingredient.SELECTED DRAWING: Figure 1

Description

The present invention relates to a novel use for treating osteoarthritis with tetrapeptide-3 GEKG or pentapeptide-3 GEKGF (use of a peptide having 4 or 5 amino acids for treating the degenerative joint disease).

Degenerative joint disease refers to a persistent lesion of articular cartilage and grows with bone tumors. Most of the bone growth occurs at the joint margin or below the cartilage. The causes of osteoarthritis are very complex, but mainly relate to the patient's age at onset. Osteoarthritis often occurs in the elderly. Occurrence of osteoarthritis is confirmed by inspection of articular cartilage wear and fibrosis, the occurrence of bone tumors (osteophytes) and / or inflammation of soft tissues.

It is known that age changes have a positive effect on cartilage lesions, although the true etiology of osteoarthritis is still unknown.

Most osteoarthritis occurs in joints that support weight or between the fingers, particularly in the distal finger joints. Joint pain and activity limitation are the main signs in the clinic. Pathological examination will check for joint deformation, joint margin swelling, and the presence of pain and joint noise when pressed.

Drugs for the treatment of osteoarthritis currently include non-steroidal anti-inflammatory drugs (eg, COX2 drug formulations), intra-articular local injection steroids, and intra-articular nutritional lubricants (ie, hyaluronic acid formulations).

Conventional Patent Document 1 describes an invention of use for treating arthritis with a tetrapeptide containing two or three alanine (Ala; A) residues (Alanine residues), for example.

Conventional Patent Document 2 describes an invention for use for treating rheumatoid arthritis with, for example, a cyclic peptide containing 8 amino acid residues and 2 binding groups.

Conventional Patent Documents 3 and 4 describe, for example, inventions for use for preventing or treating imbalance between water and minerals in tissues or organs (bones, etc.) using pentapeptide (QHNPR). Q represents glutamic acid (Gln), H represents histidine (His), N represents aspartic acid (Asn), P represents proline (Pro), and R represents arginic acid (Arg).

However, the prior art teaches and posts about treating osteoarthritis with tetrapeptide-3 GEKG / pentapeptide-3 GEKGF using high lysine (Lys; K) and threonine (Thr; T). It has not been.

US Patent Application No. US4528133 US Patent Application No. US 6034057 US Patent Application No. US65859750 B2 Specification US Patent Application No. US7429448 B2 Specification

Therefore, the present inventor considered that the above-mentioned drawbacks could be improved, and as a result of intensive studies, he succeeded in applying Tetrapeptide-3 GEKG / Pentapeptide-3 GEKGF to osteoarthritis treatment. Provide new options for osteoarthritis treatment.

The present invention has been made to solve the above-described problems of the prior art. That is, an object of the present invention is to provide a novel use for treating osteoarthritis with tetrapeptide / pentapeptide.

Thus, the present invention specifically realizes the above-described objects and effects mainly by the following technical means.

It is an influence figure with respect to a chondrocyte DNA content with a tetrapeptide. It is an influence figure with respect to the synthesis | combination of a tetrapeptide cultured collagen tissue. It is an influence figure with respect to the synthesis | combination of the tetrapeptide of a cultured cartilage tissue proteoglycan.

The present invention is a use for treating osteoarthritis with tetrapeptide-3 GEKG / pentapeptide-3 GEKGF. In the following, the technical contents of the present invention will be described using specific specific embodiments as examples, so that those skilled in the art can easily understand the advantages and effects of the present invention from the contents described in this specification. To do. However, the present invention can be practiced or applied in addition to other different specific embodiments. Changes may be made based on the design and demand of specific embodiments of the invention without departing from the scope of the patent application scope of the invention.

The pharmaceutical composition described in the present invention also comprises a therapeutically effective amount of tetrapeptide-3 GEKG / pentapeptide-3 GEKGF and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes a solid carrier, a semi-solid carrier, and a liquid carrier, and the types and appropriate doses of the solid carrier, semi-solid carrier, and liquid carrier are those conventionally used in this technical field. It is. The pharmaceutical composition takes the form of tablets, tablets, pills, powders, or granules. The pharmaceutical composition takes the form of a jelly, a poultice, or a patch. The pharmaceutical composition takes the form of a water-soluble buffer, and the water-soluble buffer includes, but is not limited to, an aqueous solution of phosphate or citrate.

The abbreviations of the definition of biochemistry used in the present invention are lysine (Lys; K), glutamate (Glu; E), and phenylalanine (Phe; F).

The tetrapeptide-3 GEKG and hexapentapeptide-3 GEKGF described in the present invention are prepared by the methods described in the prior art, for example, TW2011129368 A1 and TW201333045 A1.

The methods for measuring SOD, NO, and MDA in synovial fluid and the methods for measuring collagen and proteoglycans in the chondrocyte matrix used in the present invention are conventional in the art.

Example 1
1. Animal model 15 healthy adult New Zealand white rabbits are divided into 3 groups. Divided into a normal control group (group A), a model control group (group B), and a peptide joint cavity injection group (group C), each group having 5 animals. They are refrained from food for 4 hours before surgery, and intramuscular injections of Ketamine (10 mg / kg) and Atropine (0.2 mg / kg) are performed. After anesthesia is effective, the animal is fixed on the operating table, an 8 cm longitudinal incision is formed in the posterior edge of the patella ligament, the skin is lifted, and the joint capsule and synovium are opened along the posterior edge of the patella The knee joint is bent 90 degrees to expose the cartilage at the lower end of the femur. Two cylindrical defects having a diameter of 3 mm and a depth of 3 mm are formed, blood clots are removed from the defect, the joint lumen is washed with sterile physiological saline, and then carefully stitched in stages. Inject penicillin anti-infective drug for 1 week after surgery. After 5 days, resume normal eating and drinking and stand on your own.

2. Experimental method Starting from the second week after the surgery, no treatment is performed on group A and group B, and 250 ppm / kg of tetrapeptide-3 GEKG is injected into the joint cavity in group C, every 7 days The drug is administered for a total of 12 weeks. 0.5 ml of saline is injected into the joint cavity and 0.2 ml of joint fluid is collected. Incision of joint capsule, removal of synovium and complete removal of articular surface of tibia, observation of degree of articular cartilage lesion with naked eye and endoscope Stain) and toluidine blue staining are performed and observed with an optical microscope.

3. Experimental results <Observation results with the naked eye>
After 12 weeks, the knee joint surface of Group A was smooth and elastic, and a small amount of joint fluid was observed in the joint cavity. The defect group of group B was partially repaired, except that a depression and a gap remained in the center of the defect. In C, all defects are completely repaired, there is no difference between the cross-sectional structure and normal joints, the surface cartilage and the cancellous bone under it are repaired equally, and ossification and repair There was no trace of.

<Histomorphological observation>
A group of joint tissues is similar in characteristics to normal joint tissues, the surface is matured with cartilage tissue, mature epiphyseal cartilage plates are arranged in order in the middle, and the lower part is cancellous bone with good ossification And the thickness of the cartilage tissue is normal. The repaired tissue of the group B defect group is mainly fibrocartilage and fiber tissue, the boundary with normal tissue is continuous, and there are many fibrocartilage tissues in the defect area of the partial specimen. However, the formation of the epiphyseal cartilage plate in the defect area is not clear, and the ossification of the lower cancellous bone is poor. The repaired tissue of group C is clearly not different from the characteristics of normal joint tissue, and the surface cartilage tissue, epiphyseal cartilage plate, and lower cancellous bone all reach normal histological morphology, The thickness of cartilage is the same as that of normal cartilage, and the boundary between cartilage and bone is not divided.

<Biochemical index test>
In contrast to group B, the articular cartilage morphology of group C is clearly improved, SOD in synovial fluid is also clearly increased, and NO and MDA are also significantly decreased.

The values of NO, SOD, and MDA in each set of synovial fluid are displayed.

Conclusion 1 As is clear from the experimental results of histomorphology, when tetrapeptide-3 GEKG is injected into the joint cavity, the damaged cartilage tissue is markedly repaired, and the therapeutic effect of osteoarthritis is sufficiently confirmed.
2. Tetrapeptide-3 GEKG clearly reduces MDA in the synovium, reduces the degree of damage due to lipid peroxidation in the joint cavity, thereby reducing damage to free radical chondrocytes and matrix, To achieve the protective effect.
3. Tetrapeptide-3 GEKG significantly increases SOD concentration in the joint lumen of rabbits, eliminates free radicals generated by inflammation, protects cartilage, and achieves inhibition and prevention of cartilage lesions.
4. Tetrapeptide-3 GEKG clearly increases the NO concentration in the joint lumen of rabbits and reduces the destruction of NO to cartilage.

(Example 2)
1. Experimental method First, a rabbit growth plate chondrocyte was obtained and continuously cultured, and a tetrapeptide-3 GEKG single action group, a collagen single action group, a tetrapeptide-3 GEKG + collagen combined action group were provided. -3 GEKG 250 ppm, collagen (100 ng / ml), and tetrapeptide-3 GEKG (250 ppm) + collagen (100 ng / ml) were added respectively, and a negative control group was provided so that the new cartilage tissue reached the bottom of the centrifuge tube After growing, transfer to a 24-well culture plate and continue culturing. The culture medium is changed every other day. After reaching the planned culture time (3 days, 7 days and 14 days), the specimen is taken out and each item is removed. Perform an inspection.

Then, histology and histochemical examination are performed. Collect specimens, perform HE staining, and perform immunohistochemistry (S-P method).

Thereafter, the DNA content of the chondrocytes is measured using the Hoechst 33258 method.

Subsequently, the chondrocyte matrix is synthesized. Apart from water, the most major components in the chondrocyte matrix are collagen and proteoglycans composed of glycosaminoglycans and core proteins. The content of collagen and proteoglycan in the new tissue is measured, and the quantitative analysis of the substrate content and metabolic status is performed.

Finally, the collagen synthesis of chondrocytes is measured by the hydroxyproline method. Since the ratio of hydroxyproline in collagen is relatively constant (accounting for about 10% of the weight of collagen), it is possible to analyze the amount of collagen produced by chondrocytes by quantifying hydroxyproline. Operate in strict accordance with the instructions in the hydroxyproline test reagent box and calculate the collagen content of each sample.

2. Experimental result 1. Effect of tetrapeptide-3 GEKG on chondrocyte proliferation. The proliferation of chondrocytes by adding tetrapeptide-3 GEKG is remarkable, and the DNA content of the chondrocytes is statistically different from the control group (see FIG. 1). From the above results, it can be seen that tetrapeptide-3 GEKG effectively promotes the proliferation of chondrocytes.
2. Effect of tetrapeptide-3 GEKG on chondrocyte matrix synthesis. Addition of tetrapeptide-3 GEKG significantly promotes the synthesis of collagen and proteoglycan, and further effectively promotes the proliferation of chondrocytes, which show statistical differences from the control group (See FIGS. 2 and 3).

As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the concrete structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included.

None

Claims (12)

  Use for preparing a drug with tetrapeptide-3 GEKG, characterized in that the drug is used for the treatment of osteoarthritis in patients.   Use for preparing a drug with tetrapeptide-3 GEKG according to claim 1, characterized in that the SOD content in the joint fluid of patients undergoing the treatment is increased and the NO and MDA content is reduced.   The use for preparing a drug with tetrapeptide-3 GEKG according to claim 1, characterized in that articular chondrocytes of the patient who has undergone the treatment proliferate.   A pharmaceutical composition comprising a therapeutically effective amount of tetrapeptide-3 GEKG and a pharmaceutically acceptable carrier.   The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition is in the form of a tablet, tablet, pill, powder, granule, jelly, poultice, patch, or water-soluble buffer.   The pharmaceutical composition according to claim 5, wherein the water-soluble buffer is an aqueous solution of phosphate or citrate.   Use for preparing a drug with pentapeptide-3 GEKGF, characterized in that the drug is used for the treatment of osteoarthritis in patients.   Use for preparing a drug with pentapeptide-3 GEKGF according to claim 7, characterized in that the SOD content in the synovial fluid of the treated patient is increased and the NO and MDA content is decreased.   8. Use for preparing a drug with pentapeptide-3 GEKGF according to claim 7, characterized in that articular chondrocytes of the patient who has undergone the treatment proliferate.   A pharmaceutical composition comprising a therapeutically effective amount of pentapeptide-3 GEKGF and a pharmaceutically acceptable carrier.   The pharmaceutical composition according to claim 10, wherein the pharmaceutical composition is in the form of a tablet, tablet, pill, powder, granule, jelly, cataplasm, patch, or water-soluble buffer.   The pharmaceutical composition according to claim 11, wherein the water-soluble buffer is an aqueous solution of phosphate or citrate.