WO2020175721A1 - Hapln1을 포함하는 연골 관련 질환 또는 증상의 예방, 개선 또는 치료용 조성물 - Google Patents
Hapln1을 포함하는 연골 관련 질환 또는 증상의 예방, 개선 또는 치료용 조성물 Download PDFInfo
- Publication number
- WO2020175721A1 WO2020175721A1 PCT/KR2019/002418 KR2019002418W WO2020175721A1 WO 2020175721 A1 WO2020175721 A1 WO 2020175721A1 KR 2019002418 W KR2019002418 W KR 2019002418W WO 2020175721 A1 WO2020175721 A1 WO 2020175721A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hapln1
- cartilage
- protein
- composition
- active ingredient
- Prior art date
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/728—Hyaluronic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
Definitions
- the present invention relates to a composition for preventing, improving, or treating cartilage-related diseases or symptoms comprising hyaluronan and proteoglycan link protein 1 (HAPLN1) as an active ingredient.
- the present invention relates to a composition for cartilage regeneration comprising HAPLN1 as an active ingredient, or a composition for preventing, improving or treating osteoarthritis.
- the present invention relates to a composition for promoting growth plate cartilage proliferation or bone length growth, or a composition for preventing, improving or preventing short stature.
- Osteoarthritis is a degenerative joint disease that stimulates joint pain, joint swelling, stiffness and reduced range of motion in patients, and causes destruction of joint cartilage and major bones. Osteoarthritis is known to develop in at least 1 out of 10 adults (Lawrence, Felson et al. 2008), and the patient's quality of life is significantly lowered due to pain associated with joint movement. Osteoarthritis was previously thought to be a common result of aging, but is now known to arise from a complex interaction of many factors including joint integrity, genetic predisposition, local inflammation, mechanical forces, and cellular and biochemical processes. Its pathogenic features include hypertrophy of the lower subchondral bone and formation of osteophytes (bone processes), and degeneration of the extracellular matrix (ECM) of articular cartilage that fills the joint.
- ECM extracellular matrix
- the thickness of cartilage in the knee joint is about 2 mm, and when the area is damaged by 1 to 4 mm2 due to trauma or disease, regeneration by natural healing is possible, but when damaged by 20 mm2, regeneration by magnetic force is difficult. It is usually accompanied by great pain.
- treatments such as embedding an artificial joint in the corresponding location are performed in order to restore joint function.
- artificial joints are constructed artificially similar to joint function to the last, and are foreign objects in the living body, so it is difficult to maintain biocompatibility.
- IGF-1 Insulin-like growth factor 1
- IGF-1 Intra-like growth factor 1
- the concentration of IGF-1 in the blood increases when the growth hormone is administered, it is determined that it works normally, and when growth hormone or IGF-1 acts on the cartilage cells, growth factors are secreted and the growth plate proliferates.
- growth hormone injection is not suitable for children whose hormone secretion occurs normally, and when used, it causes side effects such as hypothyroidism or hyperpituitary gland function, and has a problem with high cost. Accordingly, there is an urgent need to develop a material capable of inducing height growth by promoting growth of growth plate cartilage.
- Articular cartilage and growth plate cartilage correspond to hyaline cartilage among the types of cartilage.
- the extracellular matrix (ECM) of this cartilage tissue is collagen type II (type II collagen), aggrecan, hyaluronan, HAPLN1 (hyaluronan and proteoglycan link protein 1), etc. It has an aggregate structure.
- a number of aggrecans are bonded to the hyaluronic acid chain, and HAPLN1 is known to play a role of physically and chemically stabilizing the aggregate by binding the bonds more strongly.
- HAPLN1 has the effect of treating, preventing or improving cartilage-related diseases or symptoms.
- U.S. Patent Publication No. 2016/0220699 discloses, including the step of delivering an expression vector encoding one or more therapeutic polynucleotides to a joint of an individual, cartilage cells or cartilage type A method of generating cells is disclosed.
- International Patent Publication No. 2017/123951 discloses a method for producing chondrocytes or cells similar thereto, comprising the step of providing a component derived from nucleus (nucleus pulposus) to a patient with degenerative disc disease.
- nucleus nucleus pulposus
- An object of the present invention is to provide a composition for preventing, improving, or treating cartilage-related diseases or symptoms using HAPLN1.
- an object of the present invention is to provide a composition for cartilage regeneration, or a composition for preventing, improving, or treating osteoarthritis.
- an object of the present invention is to provide a composition for proliferating growth plate cartilage or promoting bone length growth, or a composition for preventing, improving or treating short stature.
- the present invention is a pharmaceutical composition for cartilage regeneration, a food composition for cartilage regeneration, or an animal for cartilage regeneration, comprising hyaluronan and proteoglycan link protein 1 (HAPLN1) as an active ingredient. It provides a feed composition for use.
- HPLN1 proteoglycan link protein 1
- the present invention provides a pharmaceutical composition, food composition, or animal feed composition for preventing, improving, or treating osteoarthritis comprising HAPLN1 as an active ingredient.
- the present invention provides a pharmaceutical composition for growth plate cartilage proliferation or for promoting bone length growth, comprising HAPLN1 as an active ingredient.
- the present invention provides a food composition for growth plate cartilage proliferation and bone length growth promotion, comprising HAPLN1 as an active ingredient.
- the present invention provides a feed composition for animals for promoting growth plate cartilage proliferation and bone length growth, comprising HAPLN1 as an active ingredient.
- the present invention provides a pharmaceutical composition for treating or preventing bone length growth disorders comprising HAPLN1 as an active ingredient.
- Bone length growth disorder of the present invention may be short stature, small certificate, dwarfism or precocious puberty.
- HAPLN1 protein has the ability to promote cartilage formation and regenerate articular cartilage, and increase the level of expression of the TGF- ⁇ receptor I protein in chondrocytes, thereby increasing the cartilage-forming ability-bearing cells, and inducing the regeneration of cartilage tissue.
- the HAPLN1 protein of the present invention is a substance that regulates signal transduction of TGF- ⁇ and can be usefully used as a pharmaceutical composition for cartilage regeneration, a food composition for cartilage regeneration, or an animal feed composition for cartilage regeneration.
- HAPLN1 protein proliferates growth plate cartilage and promotes bone length growth, so that it can be provided as a key growth therapeutic agent and adjuvant, which has fewer side effects and superior effects compared to conventional hormone drugs.
- FIG. 1 shows the results of SDS-PAGE analysis of recombinant human HAPLN1 protein obtained using Expi TM 293 cells as a protein expression system.
- Figure 2 is a result of confirming the cartilage formation ability due to repeated intraperitoneal administration of the HAPLN1 protein to the degenerated growth plate of aging mice, and Figure 2 (A) is a proteoglycan in the tissue safranin O / Fast Green FCF ) It was confirmed by staining, and FIG. 2(B) shows the presence of chondrocytes having cartilage-forming ability by immunohistochemistry.
- FIG. 3 shows the cartilage regeneration ability of HAPLN1 protein administered into the joint cavity for damaged knee joint tissue of a mouse by immunofluorescence staining.
- FIG. 4 is a result of confirming the ability of HAPLN1 protein to promote cartilage formation on human articular chondrocytes
- FIG. 4(A) is a polymerase for the expression levels of the cartilage-specific gene SOX9 and aggrecan and collagen type II, which are cartilage matrix components. It was confirmed through a polymerase chain reaction (PCR), and FIG. 4(B) shows the proteoglycan accumulated in the extracellular matrix was confirmed by safranin O/fast green FCF staining.
- FIG. 5 is a confirmation of the TGF- ⁇ signaling ability of the HAPLN1 protein to mouse articular chondrocytes
- FIG. 5(A) is a Western blot confirming the TGF- ⁇ receptor I regulatory ability of the HAPLN1 protein
- Figures 5 (B) and 5 (C) confirm the stabilization of the TGF- ⁇ receptor I of the HAPLN1 protein by polymerase chain reaction and Western blot
- Figure 5 (D) shows the cell surface TGF- ⁇ receptor I by the HAPLN1 protein. The performance improvement was confirmed by Western blot.
- FIG. 6 shows a schematic diagram summarizing the experimental group of six mice used to analyze the ability of HAPLN1 protein to improve osteoarthritis.
- FIG. 7 is a result of confirming the ability of HAPLN1 protein to improve articular cartilage tissue morphology in osteoarthritis-induced mice
- FIG. 7(A) shows the articular cartilage tissue morphology in each mouse experimental group as safranin O/Fast Green FCF (Safranin O/Fast Green FCF) was confirmed by staining
- FIG. 7(B) is a quantification of the osteoarthritis improvement ability of the HAPLN1 protein through the osteoarthritis histopathological evaluation method (OARSI score).
- the inventors of the present invention confirmed the ability to promote cartilage formation and cartilage regeneration by HAPLN1 protein in aging mice and mice with joint cartilage damage. In addition, excellent cartilage formation promoting ability and cartilage regeneration ability by HAPLN1 protein were confirmed in chondrocytes. In addition, the present invention was completed by confirming that the HAPLN1 protein selectively and stably increases the TGF- ⁇ receptor I protein of chondrocytes.
- the present invention provides a pharmaceutical composition for cartilage regeneration comprising HAPLN1 protein as an active ingredient.
- the HAPLN1 protein may promote cartilage formation and protect articular cartilage.
- the HAPLN1 protein can selectively and stably increase the TGF- ⁇ receptor I protein to increase cartilage-forming ability-bearing cells, and induce regeneration of cartilage tissue.
- the present invention provides a method of regenerating cartilage in a subject comprising administering an effective amount of HAPLN1 protein to a subject in need thereof.
- subject as used herein includes human and non-human animals.
- Non-human animals include all vertebrates, for example mammals and non-mammals, such as non-human primates, sheep, dogs, cattle, horses and the like.
- the invention provides the use of the HAPLN1 protein for cartilage regeneration.
- the present invention also provides a food or animal feed composition for cartilage regeneration comprising HAPLN1 protein as an active ingredient.
- the present invention provides a pharmaceutical composition for the prevention, improvement, or treatment of osteoarthritis comprising HAPLN1 protein as an active ingredient.
- osteoarthritis is a disease commonly referred to as degenerative arthritis, and the joint cartilage surrounding the joint surface of the bone is worn out, exposing the bone under the cartilage, and causing pain and deformation due to inflammation in the synovial membrane around the joint. It is a disease, and is interpreted as a commonly understood and used meaning in the art. Externally, a bone called an osteophyte grows at the edge of the bone, causing the joint to protrude and deform the joint.
- pain relievers are used as drug therapy to relieve pain
- NSAID nonsteroidal
- the articular cartilage tissue in which osteoarthritis develops is improved to have a shape similar to that of normal cartilage. Furthermore, when the HAPLN1 protein is used, the degree of damage to the joint surface is reduced, and the number of fibrosis and osteophyte is reduced, so that the progression of osteoarthritis can be prevented, inhibited or improved.
- the present invention provides a method of preventing, ameliorating, or treating osteoarthritis in a subject comprising administering an effective amount of HAPLN1 protein to a subject in need thereof.
- the present invention provides the use of the HAPLN1 protein for the prevention, amelioration, or treatment of osteoarthritis.
- the present invention also provides a food or animal feed composition for preventing or improving osteoarthritis comprising HAPLN1 protein as an active ingredient.
- the present invention provides a pharmaceutical composition for growth plate cartilage proliferation or bone length growth promotion comprising the HAPLN1 protein as an active ingredient.
- the HAPLN1 protein of the present invention promotes the formation of cartilage in the growth plate, enhances the growth plate activity, and strengthens the growth plate. Accordingly, HAPLN1 protein can be used for the treatment or prevention of bone length growth disorders.
- the bone length growth disorder includes, but is not limited to, short stature, small authentication, dwarfism, or precocious puberty.
- the present invention provides a method for proliferating growth plate cartilage or promoting bone length growth in a subject, comprising administering an effective amount of HAPLN1 protein to a subject in need of promoting growth plate cartilage proliferation or bone length growth. to provide.
- the present invention also provides a method of treating or preventing bone length growth disorder in a subject, comprising administering an effective amount of HAPLN1 protein to a subject in need thereof.
- the invention provides the use of the HAPLN1 protein for promoting growth plate cartilage proliferation or bone length growth.
- the present invention also provides the use of HAPLN1 protein for the treatment or prevention of bone length growth disorders.
- the present invention also provides a food or animal feed composition for growth plate cartilage proliferation and bone length growth promotion comprising HAPLN1 protein as an active ingredient.
- the present invention provides a method for regenerating cartilage tissue by treating cartilage cells with HAPLN1. Specifically, separating the chondrocytes from the subject; And it provides a method for regenerating cartilage tissue in vitro using HAPLN1, comprising the step of treating the chondrocytes with HAPLN1.
- composition of the present invention is a pharmaceutical composition, for administration, it may include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the above-described active ingredients.
- composition of the present invention is a food composition
- various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like may be contained.
- a recombinant human HAPLN1 gene to which 10 histidines are bound was synthesized, inserted into the plasmid pcDNA3.4-TOPO, and transfected into Expi TM 293 cells using a protein expression system. After 3 days of culture, the cell culture solution was collected, passed through the HisTrap column, which is a chelate column, to purify the protein, and then purified by eluting with a gradient of 20 mM to 500 mM imidazole salt using an imidazole PBS buffer solution containing 0.5 M NaCl. I did.
- TEV protease was treated to remove the bound histidine, and the added TEV protease was removed again using DynaBeads.
- the resulting HAPLN1 protein was loaded on SDS-PAGE to confirm the purified protein (Fig. 1). After confirming the degree of purification, a protein having a purity of 98% or higher was obtained, which was used for in vitro cell experiments or in vivo efficacy tests.
- Example 1 Analysis of cartilage regeneration ability by HAPLN1 protein in degenerated cartilage tissue in vivo
- HAPLN1 protein was added to phosphate buffered saline (PBS). Diluted and intraperitoneally administered daily for 2 weeks at a dose of 0.1 mg/kg, the control group was intraperitoneally administered with PBS in an equivalent manner.
- PBS phosphate buffered saline
- the growth plate of the old control degenerates and only traces of cartilage tissue can be confirmed, whereas the HAPLN1 protein was repeatedly administered intraperitoneally.
- the aging group (Old HAPLN1), it was confirmed that cartilage was formed on the degenerated growth plate (arrow head).
- the young control has cells expressing SOX9 overall in the cartilage tissue
- the aging control (Old Control) cells expressing SOX9 can be found at all. There was no. However, it was confirmed that a number of SOX9-expressing cells were found in the cartilage formation stimulation site of the aging group (Old HAPLN1) repeatedly intraperitoneally administered with the HAPLN1 protein (arrow).
- the HAPLN1 protein proliferates or regenerates growth plate cartilage, thereby promoting growth plate strengthening and bone length growth.
- Example 2 Analysis of cartilage regeneration ability by HAPLN1 protein in damaged cartilage tissue in vivo
- mice Seven-week-old male C57BL/6 mice were divided into three groups as follows.
- the normal control group Sham Control group was a sham operation for the destabilization of medial meniscus (DMM) procedure, and was reared under the existing conditions for 4 weeks after the procedure.
- Vehicle treatment group (DMM Control group) was reared under the existing conditions for 8 weeks after DMM procedure, and PBS was administered intraarticularly once a week for the last 4 weeks.
- the HAPLN1 treatment group (DMM HAPLN1 group) was reared under the existing conditions for 8 weeks after DMM procedure, and for the last 4 weeks, HAPLN1 protein was diluted in PBS at a concentration of 1 ⁇ g/mL once a week and administered intraarticularly.
- the HAPLN1 protein has an excellent effect on cartilage regeneration.
- Example 3 Analysis of the ability of HAPLN1 protein to promote cartilage formation in vitro
- HAC Human articular chondrocyte
- FBS fetal bovine serum
- Cibco penicillin/streptomycin
- DMEM/F12 Dulbecco's modified Eagle medium/F12 1:1 mixture
- NEAA amino acid
- Gibco Dulbecco's modified Eagle medium/F12 1:1 mixture
- HAC cartilage-forming ability
- RNA extraction and polymerase chain reaction were performed on the cells obtained after centrifugation to compare and analyze gene expression patterns, and the specific process is as follows.
- First-strand cDNA was synthesized from 0.1 ⁇ g of the obtained RNA using oligo-dT20 primer and SuperScript III First-Strand Synthesis Supermix (Invitrogen).
- the obtained cDNA was PCR with 200 nM primers for each gene of interest using iQ SYBR Green Supermix (Bio-rad).
- iQ SYBR Green Supermix Bio-rad
- the amplified signal was measured in real time with CFX Connect (Bio-rad), and the expression level of the gene of interest was calculated as a relative value to the respective GAPDH expression level.
- the results are shown in Fig. 4(A), and the primer sequences for each human gene used in PCR are as follows.
- the HAPLN1 protein causes HAC to increase SOX9 gene expression and at the same time increase gene expression of aggrecan (ACAN) and collagen type II (type II collagen; COL2A1).
- ACAN aggrecan
- COL2A1 collagen type II collagen
- Example 4 Analysis of the ability to regulate TGF- ⁇ signaling by HAPLN1 protein
- TGF- ⁇ receptor I T ⁇ R1
- Immature murine articular chondrocytes were isolated from the articular cartilage of both legs of a 5-day-old ICR mouse.
- the obtained iMAC was in DMEM/F12 (Gibco) medium containing 10% FBS (Gibco), 1% penicillin/streptomycin (Gibco), and 1% NEAA (Gibco) at 37°C, 5% CO 2 Cultured under conditions.
- TGF- ⁇ receptor I TGF- ⁇ receptor I
- ALK1 activin receptor-like kinase 1
- TGF- ⁇ receptor II T ⁇ R2
- Gapdh Gapdh
- T ⁇ R1 protein expression level of the TGF- ⁇ receptor I (T ⁇ R1) of iMAC was increased by the HAPLN1 protein.
- ALK1 activin receptor-like kinase 1
- T ⁇ R2 TGF- ⁇ receptor II
- TGF- ⁇ receptor I T ⁇ R1
- TGF- ⁇ receptor I (T ⁇ R1) protein level by the HAPLN1 protein shown in Example 4-1 is a result of increased stability
- protein from cells cultured for 24 hours and 72 hours under the same experimental conditions At the same time, the expression patterns of the three genes compared with the levels were compared and analyzed, and at the same time, the increase in the TGF- ⁇ receptor I (T ⁇ R1) protein level by HAPLN1 was verified in an environment where de novo synthesis was restricted.
- RNA extraction and PCR process for this is as follows.
- RNA was extracted according to the manufacturer's instructions using TRIzol (Thermo Scientific) solution.
- First-strand cDNA was synthesized from 0.1 ⁇ g of the obtained RNA using oligo-dT20 primer and SuperScript III First-Strand Synthesis Supermix (Invitrogen).
- the obtained cDNA was PCR with 200 nM primers for each gene of interest using iQ SYBR Green Supermix (Bio-rad).
- iQ SYBR Green Supermix Bio-rad
- the amplification signal was measured in real time with CFX Connect (Bio-rad), and the expression level of the gene of interest was calculated as a relative value to the expression level of each Gapdh.
- the results are shown in Fig. 5(B), and the primer sequences for each mouse gene used in PCR are as follows.
- Tgfbr1 forward 5'-GTCACTGGAGTTGTACGGCA-3' reverse 5'-GGGCTGATCCCGTTGATTTC-3'
- Acvrl1 forward 5'-CTGGGTGCTCTAGGCTTGTG-3' reverse 5'-GCCCGTAGTACAGTCGCTG-3'
- Tgfbr2 forward 5'-AACAGTGATGTCATGGCCAG-3' reverse 5'-CAGACTTCATGCGGCTTCTC-3' Gapdh forward 5'-TGGCCTTCCGTGTTCCTAC-3' reverse 5'-GAGTTGCTGTTGAAGTCGCA-3'
- HAPLN1 200 ng/mL HAPLN1 from 0.5 hours before (pre) or 0.5 hours (post) treatment with cycloheximide (CHX) during exposure to iMAC cultured at high density at the bottom of the plate by treatment with 10 ⁇ M cycloheximide (CHX).
- CHX Cycloheximide
- the plate was washed with the cells attached to the plate with PBS, and the cells were collected to extract proteins in RIPA buffer.
- Western blot was performed with the extracted lysate to confirm the protein expression levels of TGF- ⁇ receptor I (T ⁇ R1), ALK1 (activin receptor-like kinase 1), TGF- ⁇ receptor II (T ⁇ R2) and Gapdh. , The results are shown in Fig. 5(C).
- TGF- ⁇ receptor I T ⁇ R1
- ALK1 activin receptor-like kinase 1
- TGF- ⁇ receptor II T ⁇ R2
- HAPLN1 protein did not induce transcription to the TGF- ⁇ receptor I (T ⁇ R1) gene and increased the half-life of the protein, indicating that the stability of the TGF- ⁇ receptor I (T ⁇ R1) protein possessed by iMAC was increased. I can.
- TGF- ⁇ receptor I T ⁇ R1
- HAPLN1 200 ng/mL HAPLN1 was treated 0.5 hours before (pre) or 0.5 hours after (post) cycloheximide (CHX) treatment during exposure to iMAC cultured at high density at the bottom of the plate by treatment with 10 ⁇ M cycloheximide (CHX) I did.
- CHX Cycloheximide
- the plate was washed with the cells attached to it with PBS, and the cell surface protein was biotin by reacting with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific) for 2 hours. Labeled. After the reaction was terminated with 0.1 M glycine solution, cells were collected and proteins were extracted in NP-40 lysis buffer (Bioworld).
- TGF- ⁇ receptor I T ⁇ R1
- ALK1 activin receptor-like
- the HAPLN1 protein selectively and stably increases the TGF- ⁇ receptor I (T ⁇ R1) protein.
- Example 5 Analysis of osteoarthritis improvement ability by HAPLN1 protein administered in vivo
- a destabilization of medial meniscus (DMM) procedure osteoarthritis model was used as a disease model that causes osteoarthritis by cutting the medial meniscotibial ligament inside the mouse knee joint.
- NC-4W No surgery control
- articular cartilage tissue For the morphological evaluation of articular cartilage tissue, the cartilage tissue of each tissue section slide was visualized through safranin O/fast green FCF (SO/FG) staining. The observation of the stained tissue section was taken using a Ni-U (Nikon) microscope and a DS-Ri1 (Nikon) digital camera, and the results are shown in Fig. 7(A).
- Fig. 7(A) it was confirmed that the non-operated normal group (NC-4W) and the simulated normal group (SC-4W) had intact articular cartilage, compared to the mild osteoarthritis group (DC-4W). And it was confirmed that the loss of the joint surface occurred as it went to the moderate osteoarthritis group (DC-8W).
- the low-dose HAPLN1 administration group (DL-8W) and the high-dose HAPLN1 administration group (DH-8W) it appeared similar to the articular cartilage form of the normal control group, and it was confirmed that the high-dose HAPLN1 administration group (DH-8W) was almost the same as the normal control group.
- the tissue stained slide obtained from Example 5-1 was evaluated by the osteoarthritis histopathological evaluation method (OARSI score) provided by the Osteoarthritis Research Society International (OARSI).
- OARSI score provided by the Osteoarthritis Research Society International
- This evaluation method quantifies the degree of joint damage, fibrosis, and osteophyte. The higher the score, the faster the pathology of osteoarthritis progresses.
- the HAPLN1 protein is effective in preventing, improving or treating osteoarthritis.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physical Education & Sports Medicine (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Polymers & Plastics (AREA)
- Rheumatology (AREA)
- Immunology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Food Science & Technology (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Marine Sciences & Fisheries (AREA)
- Animal Husbandry (AREA)
- Dermatology (AREA)
- Molecular Biology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Fodder In General (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Materials For Medical Uses (AREA)
Abstract
Description
SOX9 | forward | 5'-AGCGAACGCACATCAAGAC-3' |
reverse | 5'-CTGTAGGCGATCTGTTGGGG-3' | |
ACAN | forward | 5'-GTGCCTATCAGGACAAGGTCT-3' |
reverse | 5'-GATGCCTTTCACCACGACTTC-3' | |
COL2A1 | forward | 5'-TGGACGCCATGAAGGTTTTCT-3' |
reverse | 5'-TGGGAGCCAGATTGTCATCTC-3' | |
GAPDH | forward | 5'-CTGGGCTACACTGAGCACC-3' |
reverse | 5'-AAGTGGTCGTTGAGGGCAATG-3' |
Tgfbr1 | forward | 5'-GTCACTGGAGTTGTACGGCA-3' |
reverse | 5'-GGGCTGATCCCGTTGATTTC-3' | |
Acvrl1 | forward | 5'-CTGGGTGCTCTAGGCTTGTG-3' |
reverse | 5'-GCCCGTAGTACAGTCGCTG-3' | |
Tgfbr2 | forward | 5'-AACAGTGATGTCATGGCCAG-3' |
reverse | 5'-CAGACTTCATGCGGCTTCTC-3' | |
Gapdh | forward | 5'-TGGCCTTCCGTGTTCCTAC-3' |
reverse | 5'-GAGTTGCTGTTGAAGTCGCA-3' |
Group 1 | NC-4W | No surgery normal control | 비시술 4주 정상대조군 | 비시술 정상군 |
Group 2 | SC-4W | Sham operation normal control | 모의시술 4주 정상대조군 | 모의시술 정상군 |
Group 3 | DC-4W | DMM 4-week control (early OA) | DMM 시술 4주 음성대조군 | 경도 골관절염군 |
Group 4 | DC-8W | DMM 8-week control (moderate OA) | DMM 시술 8주 음성대조군 | 중등도 골관절염군 |
Group 5 | DL-8W | DMM low dose HAPLN1 | DMM 시술 8주 저용량 HAPLN1 투여군 | 저용량 HAPLN1 투여군 |
Group 6 | DH-8W | DMM high dose HAPLN1 | DMM 시술 8주 고용량 HAPLN1 투여군 | 고용량 HAPLN1 투여군 |
Claims (10)
- 히알루론산과 프로테오글리칸 연결 단백질 1(hyaluronan and proteoglycan link protein 1; HAPLN1)을 유효성분으로 포함하는 연골 재생용 약학 조성물.
- 제 1항에 있어서, 상기 HAPLN1은 연골 형성을 촉진하고, 관절연골을 보호하는 것을 특징으로 하는 연골 재생용 약학 조성물.
- 제1항에 있어서, 상기 HAPLN1은 TGF-β 수용체 I 단백질을 증가시켜 연골 형성능 보유 세포를 증가시키고, 연골 조직의 재생을 유도하는 것을 특징으로 하는 연골 재생용 약학 조성물.
- HAPLN1을 유효성분으로 포함하는 연골 재생용 식품 조성물.
- HAPLN1을 유효성분으로 포함하는 연골 재생용 동물용 사료 조성물.
- HAPLN1을 유효성분으로 포함하는 골관절염의 예방 또는 치료용 약학 조성물.
- HAPLN1을 유효성분으로 포함하는 성장판 연골 증식용 또는 골길이 성장 촉진용 약학 조성물.
- HAPLN1을 유효성분으로 포함하는 골길이 성장 장애 치료 또는 예방용 약학 조성물.
- 제8항에 있어서, 상기 골길이 성장 장애가 저신장증, 소인증, 왜소증 또는 성조숙증인 것을 특징으로 하는 약학 조성물.
- 연골 세포를 대상체로부터 분리하는 단계; 및HAPLN1을 연골 세포에 처리하는 단계를 포함하는, HAPLN1을 이용하여 연골 조직을 재생시키는 방법.
Priority Applications (14)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/KR2019/002418 WO2020175721A1 (ko) | 2019-02-28 | 2019-02-28 | Hapln1을 포함하는 연골 관련 질환 또는 증상의 예방, 개선 또는 치료용 조성물 |
BR112021013538-3A BR112021013538A2 (pt) | 2019-02-28 | 2019-02-28 | Composição farmacêutica para regenerar cartilagem, composição alimentícia para regenerar cartilagem, composição alimentícia animal para regenerar cartilagem, composição farmacêutica para a prevenção ou o tratamento de osteoartrite, composição farmacêutica para a proliferação da cartilagem da placa de crescimento ou o estímulo do crescimento do comprimento ósseo, composição farmacêutica para o tratamento ou a prevenção de distúrbios de crescimento do comprimento ósseo, e método para regenerar tecidos de cartilagem com o uso de hapln1 |
AU2019431082A AU2019431082B2 (en) | 2019-02-28 | 2019-02-28 | Composition, for preventing, relieving or treating cartilage-related diseases or symptoms, comprising HAPLN1 |
US17/297,707 US20230158106A1 (en) | 2019-02-28 | 2019-02-28 | Composition for preventing, relieving or treating cartilage-related diseases or symptoms, comprising hapln1 |
SG11202105767YA SG11202105767YA (en) | 2019-02-28 | 2019-02-28 | Composition, for preventing, relieving or treating cartilage-related diseases or symptoms, comprising hapln1 |
CA3122250A CA3122250C (en) | 2019-02-28 | 2019-02-28 | Composition, for preventing, relieving or treating cartilage-related diseases or symptoms, comprising hapln1 |
JP2021549676A JP7302903B2 (ja) | 2019-02-28 | 2019-02-28 | Hapln1を含む軟骨関連疾患、あるいはその症状の予防用、改善用または治療用の組成物 |
CN201980093146.6A CN113490500A (zh) | 2019-02-28 | 2019-02-28 | 包括hapln1的用于预防、改善或治疗与软骨有关的疾病或症状的组合物 |
MX2021008327A MX2021008327A (es) | 2019-02-28 | 2019-02-28 | Composicion para prevenir, mejorar o tratar enfermedades o sintomas relacionados con el cartilago que comprende hapln1. |
EP19917174.5A EP3888664A4 (en) | 2019-02-28 | 2019-02-28 | COMPOSITION FOR THE PREVENTION, RELIEF OR TREATMENT OF DISEASES OR SYMPTOMS RELATED TO HAPLN1 |
PE2021001148A PE20230436A1 (es) | 2019-02-28 | 2019-02-28 | Composicion para prevenir, mejorar o tratar enfermedades o sintomas relacionados con el cartilago que comprende hapln1 |
IL283551A IL283551A (en) | 2019-02-28 | 2021-05-30 | A composition comprising hapln1 for the prevention, alleviation or treatment of cartilage-related diseases or symptoms |
ZA2021/03815A ZA202103815B (en) | 2019-02-28 | 2021-06-03 | Composition, for preventing, relieving or treating cartilage-related diseases or symptoms, comprising hapln1 |
CONC2021/0009034A CO2021009034A2 (es) | 2019-02-28 | 2021-07-09 | Composición para prevenir, mejorar o tratar enfermedades o síntomas relacionados con el cartílago que comprende hapln1 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/KR2019/002418 WO2020175721A1 (ko) | 2019-02-28 | 2019-02-28 | Hapln1을 포함하는 연골 관련 질환 또는 증상의 예방, 개선 또는 치료용 조성물 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2020175721A1 true WO2020175721A1 (ko) | 2020-09-03 |
Family
ID=72238712
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/KR2019/002418 WO2020175721A1 (ko) | 2019-02-28 | 2019-02-28 | Hapln1을 포함하는 연골 관련 질환 또는 증상의 예방, 개선 또는 치료용 조성물 |
Country Status (14)
Country | Link |
---|---|
US (1) | US20230158106A1 (ko) |
EP (1) | EP3888664A4 (ko) |
JP (1) | JP7302903B2 (ko) |
CN (1) | CN113490500A (ko) |
AU (1) | AU2019431082B2 (ko) |
BR (1) | BR112021013538A2 (ko) |
CA (1) | CA3122250C (ko) |
CO (1) | CO2021009034A2 (ko) |
IL (1) | IL283551A (ko) |
MX (1) | MX2021008327A (ko) |
PE (1) | PE20230436A1 (ko) |
SG (1) | SG11202105767YA (ko) |
WO (1) | WO2020175721A1 (ko) |
ZA (1) | ZA202103815B (ko) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140178988A1 (en) * | 2012-10-08 | 2014-06-26 | Biotime, Inc. | Differentiated Progeny of Clonal Progenitor Cell Lines |
WO2014130411A1 (en) * | 2013-02-22 | 2014-08-28 | Emory University | Tgf-beta enhancing compositions for cartilage repair and methods related thereto |
US20160220699A1 (en) | 2013-09-09 | 2016-08-04 | Figene, Llc | Gene therapy for the regeneration of chondrocytes or cartilage type cells |
WO2017123951A1 (en) | 2016-01-14 | 2017-07-20 | Spinacyte, Llc | Cellular blend for the regeneration of chondrocytes or cartilage type cells |
KR20190024727A (ko) * | 2017-08-29 | 2019-03-08 | 중앙대학교 산학협력단 | Hapln1을 유효성분으로 함유하는 연골 재생용 조성물 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2013527834A (ja) | 2010-03-29 | 2013-07-04 | マサチューセッツ インスティテュート オブ テクノロジー | 抗炎症因子 |
US20180148494A1 (en) * | 2015-01-07 | 2018-05-31 | TheAchon | Soluble fgfr3 decoys for treating skeletal growth disorders |
AU2017403257B2 (en) * | 2017-03-06 | 2021-09-30 | Haplnscience inc. | Composition for skin aging measurement, prevention, or alleviation, using HAPLN1 |
-
2019
- 2019-02-28 SG SG11202105767YA patent/SG11202105767YA/en unknown
- 2019-02-28 JP JP2021549676A patent/JP7302903B2/ja active Active
- 2019-02-28 AU AU2019431082A patent/AU2019431082B2/en active Active
- 2019-02-28 BR BR112021013538-3A patent/BR112021013538A2/pt unknown
- 2019-02-28 US US17/297,707 patent/US20230158106A1/en active Pending
- 2019-02-28 WO PCT/KR2019/002418 patent/WO2020175721A1/ko active Application Filing
- 2019-02-28 CA CA3122250A patent/CA3122250C/en active Active
- 2019-02-28 EP EP19917174.5A patent/EP3888664A4/en active Pending
- 2019-02-28 PE PE2021001148A patent/PE20230436A1/es unknown
- 2019-02-28 MX MX2021008327A patent/MX2021008327A/es unknown
- 2019-02-28 CN CN201980093146.6A patent/CN113490500A/zh active Pending
-
2021
- 2021-05-30 IL IL283551A patent/IL283551A/en unknown
- 2021-06-03 ZA ZA2021/03815A patent/ZA202103815B/en unknown
- 2021-07-09 CO CONC2021/0009034A patent/CO2021009034A2/es unknown
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140178988A1 (en) * | 2012-10-08 | 2014-06-26 | Biotime, Inc. | Differentiated Progeny of Clonal Progenitor Cell Lines |
WO2014130411A1 (en) * | 2013-02-22 | 2014-08-28 | Emory University | Tgf-beta enhancing compositions for cartilage repair and methods related thereto |
US20160220699A1 (en) | 2013-09-09 | 2016-08-04 | Figene, Llc | Gene therapy for the regeneration of chondrocytes or cartilage type cells |
WO2017123951A1 (en) | 2016-01-14 | 2017-07-20 | Spinacyte, Llc | Cellular blend for the regeneration of chondrocytes or cartilage type cells |
KR20190024727A (ko) * | 2017-08-29 | 2019-03-08 | 중앙대학교 산학협력단 | Hapln1을 유효성분으로 함유하는 연골 재생용 조성물 |
Non-Patent Citations (1)
Title |
---|
URANO ET AL.: "Single-nucleotide polymorphism in the hyaluronan and proteoglycan link protein 1 (HAPLN1) gene is associated with spinal osteophyte formation and disc degeneration in Japanese women", EUROPEAN SPINE JOURNAL, vol. 20, 2011, pages 572 - 577, XP019891028, DOI: 10.1007/s00586-010-1598-0 * |
Also Published As
Publication number | Publication date |
---|---|
AU2019431082A1 (en) | 2021-07-01 |
JP7302903B2 (ja) | 2023-07-04 |
IL283551A (en) | 2021-07-29 |
MX2021008327A (es) | 2021-08-11 |
EP3888664A4 (en) | 2021-12-15 |
US20230158106A1 (en) | 2023-05-25 |
CN113490500A (zh) | 2021-10-08 |
ZA202103815B (en) | 2022-09-28 |
SG11202105767YA (en) | 2021-06-29 |
BR112021013538A2 (pt) | 2021-09-14 |
JP2022522144A (ja) | 2022-04-14 |
EP3888664A1 (en) | 2021-10-06 |
AU2019431082B2 (en) | 2022-12-15 |
CO2021009034A2 (es) | 2021-07-30 |
PE20230436A1 (es) | 2023-03-08 |
CA3122250A1 (en) | 2020-09-03 |
CA3122250C (en) | 2023-12-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR101706642B1 (ko) | 연골세포로 분화되고 있는 줄기세포로부터 추출된 엑소좀을 포함하는 연골세포 분화 유도 또는 연골조직 재생용 조성물 | |
Armiento et al. | Articular fibrocartilage-Why does hyaline cartilage fail to repair? | |
KR20200104831A (ko) | Hapln1을 포함하는 연골 관련 질환 또는 증상의 예방, 개선 또는 치료용 조성물 | |
WO2013009102A2 (ko) | 콜라겐, 히알루론산 유도체 및 포유류의 탯줄 유래 줄기세포를 포함하는 연골세포치료제 | |
WO2019112177A1 (ko) | 줄기세포 유래 엑소좀 함유 배양액을 유효성분으로 포함하는 관절염의 예방 또는 치료용 조성물 | |
WO2015194753A1 (ko) | Cpne7 단백질을 포함하는 비치계 중간엽 줄기세포의 상아모세포로의 분화방법, 조성물 및 이를 이용한 치수조직 재생 및 상아질 지각과민증 치료용 약학적 조성물 | |
US11213572B2 (en) | Cartilage regeneration composition containing HAPLN1 as active ingredient | |
US20020009432A1 (en) | Therapeutic agent for cartilaginous diseases | |
WO2016144146A1 (ko) | 관절염 예방 또는 치료용 약학적 조성물 | |
WO2020175721A1 (ko) | Hapln1을 포함하는 연골 관련 질환 또는 증상의 예방, 개선 또는 치료용 조성물 | |
Ruscitto et al. | Lgr5-expressing secretory cells form a Wnt inhibitory niche in cartilage critical for chondrocyte identity | |
JP2021080184A (ja) | 変形性関節症の予防又は治療剤、及び変形性関節症の予防又は治療用医薬組成物 | |
WO2022015106A1 (ko) | 종양괴사인자-자극된 유전자 6을 발현하는 중간엽 줄기세포를 포함하는 골관절염의 예방 또는 치료용 조성물 | |
WO2016126122A2 (ko) | 연골세포로 분화되고 있는 줄기세포로부터 추출된 엑소좀을 포함하는 연골세포 분화 유도 또는 연골조직 재생용 조성물 | |
RU2788162C2 (ru) | Композиция для профилактики, облегчения или лечения заболеваний или симптомов, связанных с хрящами, включающая hapln1 | |
WO2019045451A1 (ko) | Hapln1을 유효성분으로 함유하는 연골 재생용 조성물 | |
US20240139376A1 (en) | Reconstitution of extracellular matrixes for musculoskeletal joint tissue repair using biomimetic biologic and synthetic factors | |
WO2022025493A1 (ko) | 중간엽 줄기세포를 포함하는 근육질환 치료 또는 예방용 약학적 조성물 | |
WO2021210812A1 (ko) | 섬유질환 예방 또는 치료용 재조합 융합 단백질 | |
WO2024122723A1 (ko) | 연골 재생용 펩타이드 및 이의 용도 | |
CA2197869C (en) | Therapeutic agent for cartilaginous diseases | |
JP3737532B2 (ja) | 軟骨障害治療剤 | |
WO2022191341A1 (ko) | 줄기세포로부터 유래한 엑소좀을 포함하는 관절염 완화 또는 치료용 약학적 조성물 | |
WO2024122720A1 (ko) | 연골 재생용 펩타이드 및 이의 용도 | |
WO2022119061A1 (ko) | 불멸화 고양이 줄기세포 또는 이의 용도 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 19917174 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 283551 Country of ref document: IL |
|
ENP | Entry into the national phase |
Ref document number: 3122250 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2019431082 Country of ref document: AU Date of ref document: 20190228 Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2019917174 Country of ref document: EP Effective date: 20210629 |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112021013538 Country of ref document: BR |
|
ENP | Entry into the national phase |
Ref document number: 2021549676 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 112021013538 Country of ref document: BR Kind code of ref document: A2 Effective date: 20210708 |