CN107670021B

CN107670021B - Application of Wushengtai KTTKS in treating degenerative arthritis

Info

Publication number: CN107670021B
Application number: CN201610623696.2A
Authority: CN
Inventors: 庄国升
Original assignee: Ahb Lab Biotechnology Co ltd
Current assignee: Ahb Lab Biotechnology Co ltd
Priority date: 2016-08-02
Filing date: 2016-08-02
Publication date: 2020-09-18
Anticipated expiration: 2036-08-02
Also published as: CN107670021A

Abstract

The invention discloses application of pentapeptide/hexapeptide KTTKS (F) in treating degenerative arthritis.

Description

Application of Wushengtai KTTKS in treating degenerative arthritis

Technical Field

The invention relates to a new application of pentapeptide/hexapeptide KTTKS (F) in treating degenerative arthritis.

Background

Degenerative joint disease (degenerative joint disease) refers to a persistent lesion of articular cartilage and is accompanied by proliferation of neoplastic bone, with the proliferative bone mostly occurring at the joint margin or under the cartilage. The causes of degenerative arthritis are rather complex, but are mainly related to the age of the patient at the time of onset. Degenerative arthritis often occurs in older adults. The confirmation of the development of degenerative arthritis is based on the detection of wear and fibrosis of articular cartilage, the development of neoplastic bone (spur) and/or inflammation of soft tissues. Although age changes are known to actually affect cartilage changes, the true pathogenesis of degenerative arthritis remains unknown to date.

Degenerative arthritis occurs mostly in the joints or interphalangeal joints of the fingers bearing body weight, especially the distal knuckles, where joint pain and limitation of motion are the major clinical signs. Pathological examination often found joint deformity and joint edge swelling and pressure pain and even joint squeal.

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

US 4528133 discloses the use of certain tetrapeptides comprising 2 or 3 alanine (Ala; A) residues for the treatment of arthritis.

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

US 6589750B 2 and US 7429448B 2 disclose the use of the pentapeptide QHNPR for the prevention or treatment of water-mineral imbalance of a tissue or organ, such as bone, wherein Q represents glutamine (Gln); h represents histidine (His); n represents asparaginic acid (Asn); p represents proline (Pro); and R represents arginine (Arg).

None of the prior art teaches or suggests: peptides enriched with lysine (Lys; K) and threonine (Thr; T) may be used to treat degenerative arthritis.

Therefore, the present inventors have conducted extensive studies on the existing problems and, with the help of many years of research and development and manufacturing experience in the related industries, have succeeded in developing the five/six peptide kttks (f) useful for the treatment of degenerative arthritis through continuous improvement and trial, thereby providing a new option for the treatment of degenerative arthritis.

Disclosure of Invention

Therefore, the main object of the present invention is to provide the use of the penta/hexapeptide kttks (f) in the treatment of degenerative arthritis.

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

In order to enable the applicant to further appreciate the structure, features and other objects of the present invention, preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, which will enable one skilled in the art to practice the invention.

Drawings

FIG. 1 shows the effect of pentapeptide on DNA content of chondrocytes;

FIG. 2 shows the effect of pentapeptide on collagen synthesis in cultured cartilage tissue;

FIG. 3 shows the effect of pentapeptide on the synthesis of polysaccharide protein in cultured cartilage tissue.

Detailed Description

The invention discloses application of pentapeptide/hexapeptide KTTKS (F) in treating degenerative arthritis. The present invention is described in terms of specific embodiments, which will enable those skilled in the art to readily understand the advantages and capabilities of the present invention as disclosed herein. However, the invention is capable of other and different embodiments and of being practiced or being carried out in various ways. Changes may be made in the design and requirements of particular embodiments of the invention without departing from the scope of the claims.

The invention also discloses a pharmaceutical composition, which comprises a therapeutically effective amount of the pentapeptide/hexapeptide KTTKS (F) and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers include solid carriers, semi-solid carriers and liquid carriers, wherein the types and appropriate amounts of the solid carriers, semi-solid carriers and liquid carriers are well known and conventional to those skilled in the art. The pharmaceutical composition may be in the form of a lozenge, 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 an aqueous buffer, including but not limited to an aqueous solution of phosphate or citrate.

Furthermore, the present invention uses the abbreviation defined biochemically: lysine (Lys; K); threonine (Thr; T); serine (Ser; S); and phenylalanine (Phe; F).

The pentapeptide KTTKS (shown in SEQ ID NO. 1) and the hexapeptide KTTKSF (shown in SEQ ID NO. 2) disclosed in the present invention can be prepared according to the methods described in the prior art, such as TW 201129368A 1 and TW 201333045A 1.

The methods for measuring SOD, NO and MDA in synovial fluid and the methods for measuring collagen and proteoglycan in chondrocyte matrix used in the present invention are well known and commonly used by those skilled in the art.

Detailed description of the preferred embodiments

Example 1

First, animal model

15 healthy adult New Zealand white rabbits were randomized into 3 groups: normal control group (group a), model control group (group B) and victory articular cavity injection group (group C), each group containing 5 peptides. A4-hour preoperative fasting was performed, and Ketamine (10mg/kg) and Atropine (0.2mg/kg) were injected intramuscularly. After the anesthesia takes effect, the animal is fixed on an operating table, a 8cm longitudinal incision is made at the back edge of the ligament of the patella, the skin is lifted, the joint capsule and the synovial membrane are opened along the back edge of the patella, the knee joint is bent by 90 degrees, so that the cartilage at the lower end of the femur is exposed, 2 cylindrical defects with the diameter of 3mm and the depth of 3mm are made, the blood coagulation at the defect part is eliminated, and the joint inner cavity is cleaned by sterile normal saline and then is carefully sutured in a layered mode. Injection of penicillin after operation can resist infection for a week. After 5 days, the food can be eaten normally and can stand independently.

Second, Experimental methods

Starting at 2 weeks after surgery, groups A and B were not treated and group C was injected 250ppm/kg joint cavity with pentapeptide KTTKS once every 7 days for 12 weeks. The joint cavity was injected with 0.5ml of physiological saline, and 0.2ml of joint fluid was collected. The joint capsule is incised, the synovium is taken out, the tibial articular surface is completely taken out, the pathological change degree of the articular cartilage is observed under naked eyes and a surgical mirror, and the Hematoxylin-Eosin (HE Stain, Hematoxylin and Eosin Stain) and toluidine blue staining optical microscope observation is respectively carried out after the section.

Third, experimental results

Visual observation of the results

After 12 weeks, the knee joints in group A had smooth and elastic surfaces, and a little joint fluid was visible in the joint cavity; the group B defect group is only partially repaired, but a recess and a gap are remained in the center of the defect; all defects of the group C are completely repaired, the section structure is not different from that of a normal joint, the surface cartilage and the lower cancellous bone are completely repaired, and no ossification and repair trace is found.

Histological observation

The joint tissue of the group A has the same characteristics as the normal joint tissue, the surface is mature cartilage tissue, the middle is a mature epiphyseal plate which is arranged orderly, the lower part is cancellous bone with good ossification, and the thickness of the cartilage tissue is normal; the repair tissues of the B group of defect groups mainly comprise fiber-like cartilage and fiber tissues which are continuous with the interface of normal tissues, and a part of specimen defect areas are filled with more fibrous cartilage tissues, but no obvious epiphyseal plate is formed in the defect areas and the lower cancellous bone is poorly ossified; the characteristics of the group C repaired tissues are not obviously different from those of normal joint tissues, the surface cartilage tissues, the epiphyseal plate and the lower cancellous bone reach normal histological morphology, the thickness of the new cartilage is the same as that of the normal cartilage, and the boundary between the cartilage and the bone cannot be distinguished.

Biochemical index detection

Compared with group B, the morphology of articular cartilage in group C is obviously improved, SOD in the joint fluid is obviously increased, and NO and MDA are obviously reduced, and the results are shown in Table 1.

TABLE 1 values of NO, SOD and MDA in the synovial fluid of each group

Group of	NO(c/mol·L^-1)	SOD(c/mol·L^-1)	MDA(c/mol·L^-1)
				A	51.23±2.55	6.52±1.29	15.56±3.04
B	120.55±15.28	15.19±1.48	24.21±3.13
				C	25.11±3.65	6.18±2.17	15.23±5.62

Conclusion

1. The result of the histomorphology experiment shows that: the injection of the Wushengtai KTTKS into the joint cavity can obviously repair the damaged cartilage tissue, and has very definite treatment effect on the degenerative arthritis.

2. The pentapeptide KTTKS remarkably reduces MDA in synovial membrane and reduces the degree of lipid peroxidation injury in joint cavity, thereby reducing the injury of free radicals to chondrocytes and matrix and achieving the effect of protecting articular cartilage.

3. The Wushengtai KTTKS can obviously improve the SOD concentration in the inner cavity of the rabbit joint and eliminate free radicals generated by inflammation, thereby protecting cartilage and inhibiting and preventing cartilage pathological changes.

4. The Wushengtai KTTKS obviously improves the concentration of NO in the cavity of the rabbit joint, thereby reducing the damage of NO to cartilage.

Example 2

First, experiment method

Firstly, cartilage cells of a rabbit growth plate are taken for continuous culture, a pentapeptide KTTKS independent action group, a collagen independent action group and a pentapeptide KTTKS + collagen combined action group are arranged, 250ppm of pentapeptide KTTKS, 100ng/ml of collagen and 250ppm of pentapeptide KTTKS plus 100ng/ml of collagen are respectively added, a negative control group is arranged, after the newly-born cartilage tissue grows over the bottom of a centrifuge tube, the newly-born cartilage tissue is transferred into a 24-hole culture plate for continuous culture, culture media are replaced every other day, and after the preset culture time (3, 7 and 14 days) is reached, samples are collected for various detections.

Next, histological and histochemical examinations were carried out: the specimens were collected and examined by HE staining and immunohistochemistry (S-P method).

Thereafter, the DNA content of chondrocytes was measured by the Hoechst 33258 method.

Then, chondrocyte matrix synthesis was performed: in addition to water, the most important components in the chondrocyte matrix are collagen and proteoglycan composed of glycosaminoglycan and core protein. And (3) quantitatively analyzing the matrix content and the metabolic condition by measuring the content of collagen and proteoglycan in the new tissues.

Finally, the collagen synthesis of the chondrocytes was determined by hydroxyproline assay: since the proportion of hydroxyproline in collagen is relatively constant (about 10% by weight of collagen), chondrocyte collagen production can be analyzed by quantifying hydroxyproline. Strictly operating according to the specification of the hydroxyproline detection kit, and calculating the collagen content of each sample.

Second, experimental results

1. Effect of pentapeptide KTTKS on chondrocyte proliferation: the addition of pentapeptide KTTKS was significant in chondrocyte proliferation, wherein the DNA content of chondrocytes was statistically different from that of the control group (FIG. 1), and the result shows that the pentapeptide KTTKS can effectively promote chondrocyte proliferation.

2. Effect of pentapeptide KTTKS on chondrocyte matrix synthesis: the addition of the pentapeptide KTTKS significantly promoted the synthesis of collagen and proteoglycan and further effectively promoted the proliferation of chondrocytes, which was statistically different from the control group (fig. 2 and 3).

Therefore, the invention is an excellent innovation, which can effectively solve the existing problems and greatly improve the treatment effect, and the same or similar product creation or public use is not found in the same technical field, and the efficacy is improved. Therefore, the present invention has been made in accordance with the requirements of the invention and the related "novelty" and "creativity", and the invention is filed by law.

Claims

1. Use of pentapeptide KTTKS in the manufacture of a medicament for the treatment of degenerative arthritis in a patient wherein the treated patient has an elevated level of SOD and a reduced level of NO and MDA in the synovial fluid.

2. The use of claim 1, wherein the treated patient has articular chondrocyte proliferation.

3. The use of claim 1, wherein the medicament is in the form of a lozenge, tablet, pill, powder, granule, gel, paste, patch, or water-soluble buffer.

4. The use of claim 3, wherein the aqueous buffer is an aqueous solution of phosphate or citrate.