CA3157954A1 - Pharmaceutical composition, comprising recombinant stabilized galectin 9 protein, for prevention or treatment of rheumatoid arthritis and bone disease - Google Patents
Pharmaceutical composition, comprising recombinant stabilized galectin 9 protein, for prevention or treatment of rheumatoid arthritis and bone disease Download PDFInfo
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- CA3157954A1 CA3157954A1 CA3157954A CA3157954A CA3157954A1 CA 3157954 A1 CA3157954 A1 CA 3157954A1 CA 3157954 A CA3157954 A CA 3157954A CA 3157954 A CA3157954 A CA 3157954A CA 3157954 A1 CA3157954 A1 CA 3157954A1
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- 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
- A61K38/1732—Lectins
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- 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
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4726—Lectins
Abstract
The present invention relates to a pharmaceutical composition including a recombinant stabilized galectin 9 protein for prevention or treatment of a bone disease, particularly relates to a pharmaceutical composition for prevention or treatment of a bone disease including rheumatoid arthritis, which includes a recombinant stabilized galectin 9 protein in which amino acids of link peptides are deleted and amino acids of C-terminal domains (CCRD) are deleted or substituted, as an active ingredient.
Description
[ Descr i pti on]
[Title of Invention]
PHARMACEUTI CAL COMPOSI TI ON, COMPRI SI NG RECOMBI NANT
STAB! LI ZED GALECTI N 9 PROTEI N, FOR PREVENTI ON OR
TREATMENT OF RHEUMATOI D ARTHRI TI S AND BONE DI SEASE
[Techni cal Fi el d]
The present i nventi on rel at es to a recombi nant stabilized gal ecti n 9 protein and the use thereof, and speci f i cal I y rel at es to a pharmaceuti cal composi ti on, compri si ng recombi nant stabilized gal ecti n 9 protei n, for pr event i on or treatment of rheumatoi d art hritis and bone di sease.
[ Background Art]
It has been found that there are ani mal I ecti ns that specifically recognize sugar cqains -laving a 13-gal act osi di c structure in living bodi es, and at least 14 genes have been i dent i f i ed so far. Gal ecti ns are cl assi f i ed i nto prototype, chi meras, and tandem repeats based on the structures thereof.
Gal ecti n 9, one of the tandem repeat gal ecti ns, includes two sugar chain recogni ti on sites ( carbohydrate recogni ti on domai ns: CRD) and a I i nk pepti de regi on connecting the sites and has N-termi nal domain ( N -Terminal Carbohydrate Recognition Domain; NCRD) and a C-termi nal domain ( C- t er mi nal Carbohydrate Recogni ti on Domai n; CCRD) connected by the I i nk pepti de regi on, and various activities have reported so far. Regarding T
cells, gal ecti n 9 binds to Ti m-3 to induce apoptosi s of Ti m-3- posi ti ve Th1 cells, and i nhi bits excessive Th1 responses to suppress aut oi mmune i nf I ammat i on. I n addi ti on, gal ecti n 9 reduces Th17 cell s that are one of causes or exacerbati on factors of van i ous i ntractabl e di seases such as autoi mmune di seases, al I ergi es, and cancers expressed by Tim-3.
On the other hand, gal ecti n 9 enhances i mmuni ty i n some cases. Gal ecti n 9 bi nds to Ti m-3 on monocyt es or dendritic cells to activate the cells and promote the pr oduct i on of inflammatory cyt oki nes.
In addi ti on, the i nt er act i on of gal ecti n 9 and Ti m-3 in macrophages enhances i mmuni ty to excl ude Mycobacteri um tubercul osi s.
I n addi ti on, evi dence suggest i ng the exi stence of a genet i c pol ymor phi sm among the gal ecti n 9 genes cl oned from human cel I s or ti ssues is conf i rmed, and the recombi nant gal ecti n 9 produced usi ng E. col i as a host i s known to have an effect of i nduci ng metastasi s i nhi bi ti on and regressi on of cancer and an effect of i nduci ng apoptosi s of act i vat ed T cell s, part i cul ar I y CD4- posi ti ve T cel Is which are the cause of an excessive i mmune response, by di rect act i on on tumor cel I s ( act i vat i on to i nduce i nt ercel I ul ar adhesi on between tumor cel I s and apoptosi s) and act i on through the i mmune system. I n addi ti on, a t her apeut i c effect of human i mmunodef i ci ency virus ( HI V) infection, a therapeutic effect of at opy and asthma, and a therapeutic effect of type 1 diabetes are known.
I n usi ng gal ecti n 9 as an actual therapeuti c agent, in order to solve the probl ems of 1) protease sensitivity; 2) low solubility; and 3) I ow yi el d, a study of cleaving the linker pepti de of gal ecti n 9 to produce a gal ect i n 9 van i ant ( G9Nul I ; Non- Patent Document 1) with proteol yti c enzyme resistance and the like is continued, and an effect such as KRAS mutant col on carci noma ant it umor act i vi ty ( Non- Pat ent Document 2) i s known.
Meanwhi I e, a bone i s a dynami c ti ssue that undergoes conti nuous removal and reconstructi on through bone
[Title of Invention]
PHARMACEUTI CAL COMPOSI TI ON, COMPRI SI NG RECOMBI NANT
STAB! LI ZED GALECTI N 9 PROTEI N, FOR PREVENTI ON OR
TREATMENT OF RHEUMATOI D ARTHRI TI S AND BONE DI SEASE
[Techni cal Fi el d]
The present i nventi on rel at es to a recombi nant stabilized gal ecti n 9 protein and the use thereof, and speci f i cal I y rel at es to a pharmaceuti cal composi ti on, compri si ng recombi nant stabilized gal ecti n 9 protei n, for pr event i on or treatment of rheumatoi d art hritis and bone di sease.
[ Background Art]
It has been found that there are ani mal I ecti ns that specifically recognize sugar cqains -laving a 13-gal act osi di c structure in living bodi es, and at least 14 genes have been i dent i f i ed so far. Gal ecti ns are cl assi f i ed i nto prototype, chi meras, and tandem repeats based on the structures thereof.
Gal ecti n 9, one of the tandem repeat gal ecti ns, includes two sugar chain recogni ti on sites ( carbohydrate recogni ti on domai ns: CRD) and a I i nk pepti de regi on connecting the sites and has N-termi nal domain ( N -Terminal Carbohydrate Recognition Domain; NCRD) and a C-termi nal domain ( C- t er mi nal Carbohydrate Recogni ti on Domai n; CCRD) connected by the I i nk pepti de regi on, and various activities have reported so far. Regarding T
cells, gal ecti n 9 binds to Ti m-3 to induce apoptosi s of Ti m-3- posi ti ve Th1 cells, and i nhi bits excessive Th1 responses to suppress aut oi mmune i nf I ammat i on. I n addi ti on, gal ecti n 9 reduces Th17 cell s that are one of causes or exacerbati on factors of van i ous i ntractabl e di seases such as autoi mmune di seases, al I ergi es, and cancers expressed by Tim-3.
On the other hand, gal ecti n 9 enhances i mmuni ty i n some cases. Gal ecti n 9 bi nds to Ti m-3 on monocyt es or dendritic cells to activate the cells and promote the pr oduct i on of inflammatory cyt oki nes.
In addi ti on, the i nt er act i on of gal ecti n 9 and Ti m-3 in macrophages enhances i mmuni ty to excl ude Mycobacteri um tubercul osi s.
I n addi ti on, evi dence suggest i ng the exi stence of a genet i c pol ymor phi sm among the gal ecti n 9 genes cl oned from human cel I s or ti ssues is conf i rmed, and the recombi nant gal ecti n 9 produced usi ng E. col i as a host i s known to have an effect of i nduci ng metastasi s i nhi bi ti on and regressi on of cancer and an effect of i nduci ng apoptosi s of act i vat ed T cell s, part i cul ar I y CD4- posi ti ve T cel Is which are the cause of an excessive i mmune response, by di rect act i on on tumor cel I s ( act i vat i on to i nduce i nt ercel I ul ar adhesi on between tumor cel I s and apoptosi s) and act i on through the i mmune system. I n addi ti on, a t her apeut i c effect of human i mmunodef i ci ency virus ( HI V) infection, a therapeutic effect of at opy and asthma, and a therapeutic effect of type 1 diabetes are known.
I n usi ng gal ecti n 9 as an actual therapeuti c agent, in order to solve the probl ems of 1) protease sensitivity; 2) low solubility; and 3) I ow yi el d, a study of cleaving the linker pepti de of gal ecti n 9 to produce a gal ect i n 9 van i ant ( G9Nul I ; Non- Patent Document 1) with proteol yti c enzyme resistance and the like is continued, and an effect such as KRAS mutant col on carci noma ant it umor act i vi ty ( Non- Pat ent Document 2) i s known.
Meanwhi I e, a bone i s a dynami c ti ssue that undergoes conti nuous removal and reconstructi on through bone
2 resorpti on and f ormati on. These processes are carri ed out by two types of cel I s: osteocl asts and osteobl asts, respectively. Speci f i cal I y, ost eobl asts are responsi bi e for bone f ormat i on, and osteocl asts are responsi bl e for bone removal . I n a normal envi ronment, bone homeostasi s is maintained by the balanced activity of the two cells.
However, homeost asi s can be di sr upt ed by abnormal hyperactivity of these two types of cells, particularly osteocl asts, resul ti ng i n a bone di sease such as ar t hr i ti s, ost eopor osi s, per i pr ost het i c ost eol ysi s, and Paget' s di sease. Therefore, accurate r egul at i on of ost eocl ast differentiation and functions is important in the prevent i on and treatment of a bone di sease.
For example, as a drug therapy for treating osteoporosi s, as a drug that prevents a decrease i n bone mass or i ncr eases bone mass by promoti ng bone f ormat i on or suppressing bone resorption, there are bone resorption i nhi bi tors such as cal ci um preparati ons, vi tami n D
preparati ons, bi sphosphonate preparati ons, estrogen agoni sts/ ant agoni sts, and estrogen preparati ons, and ost eogenesi s promoters such as ten i par at i de i nj ect i on that i s a parathyroi d hormone preparati on.
Also, among arthritis, rheumatoid arthritis is a typi cal chroni c autoi mmune di sease and i s a di sease caused by i nf I ammati on i n a synovi al ti ssue that surrounds a joint. The rheumatoid arthritis is a di sease that can occur i n any j oi nt where the synovi al membrane exi sts and that i nvades van i ous organs throughout the body, and i s a chroni c i nf I ammatory di sease that destroys articular tissues to cause severe joint disorders and I ead to premature death. I n some severe cases, though the cases are rare, ti ssues other than j oi nts, for exampl e, the I ungs, the heart, the eyes, the gast roi nt est i nal tract, the ski n, and the ki dneys may be i nvol ved.
However, homeost asi s can be di sr upt ed by abnormal hyperactivity of these two types of cells, particularly osteocl asts, resul ti ng i n a bone di sease such as ar t hr i ti s, ost eopor osi s, per i pr ost het i c ost eol ysi s, and Paget' s di sease. Therefore, accurate r egul at i on of ost eocl ast differentiation and functions is important in the prevent i on and treatment of a bone di sease.
For example, as a drug therapy for treating osteoporosi s, as a drug that prevents a decrease i n bone mass or i ncr eases bone mass by promoti ng bone f ormat i on or suppressing bone resorption, there are bone resorption i nhi bi tors such as cal ci um preparati ons, vi tami n D
preparati ons, bi sphosphonate preparati ons, estrogen agoni sts/ ant agoni sts, and estrogen preparati ons, and ost eogenesi s promoters such as ten i par at i de i nj ect i on that i s a parathyroi d hormone preparati on.
Also, among arthritis, rheumatoid arthritis is a typi cal chroni c autoi mmune di sease and i s a di sease caused by i nf I ammati on i n a synovi al ti ssue that surrounds a joint. The rheumatoid arthritis is a di sease that can occur i n any j oi nt where the synovi al membrane exi sts and that i nvades van i ous organs throughout the body, and i s a chroni c i nf I ammatory di sease that destroys articular tissues to cause severe joint disorders and I ead to premature death. I n some severe cases, though the cases are rare, ti ssues other than j oi nts, for exampl e, the I ungs, the heart, the eyes, the gast roi nt est i nal tract, the ski n, and the ki dneys may be i nvol ved.
3 Moreover, general 1 y, i nf I ammat ory medi at ors that are secreted in the body in large amounts adversely affect bone met abol i sm, thereby i ncreasi ng the r i sk of osteoporosis and fractures. In reality, it is conf i r med that the i nci dence of ost eoporosi s in r heumat oi d arthritis patients is about 15% to 20%.
Drug therapies for treating rheumatoid arthritis i ncl ude pri mary drugs such as nonsteroi dal anti -i nf I ammat ory drugs and st er oi d preparat i ons, whi ch are a ki nd of hormone, and secondary drugs that suppress rheumatoid arthritis itself by affecting the immune system of the body, and are usual 1 y used for a long period of time, thereby requiring attention to side effects. In particular, steroid preparations may be abused because good effects are exhi bi t ed i mmedi at el y, but pr obl ems of si de effects are ser i ous. The long- t erm use thereof may be a r i sk factor for ost eoporosi s.
Accor di ngl y, the present i nvent ors have made efforts to devel op a new t her apeut i c agent havi ng an effective therapeutic effect while minimizing the side effects of a therapeutic agent for arthritis, including rheumatoid arthritis, and/or osteoporosis. As a result, it is conf i rmed that stabilized gal ect i n 9 protein in which ami no aci ds of the C- t ermi nal domai n ( CCRD) of the two sugar chain recognition sites and of link peptides of the existing wild-type gal ect i n 9 are del et ed and substituted have an effect of suppr essi ng Thl and Th17 differentiation and i nduci ng f i brobl ast - I i ke synovi ocyt es ( FLS) apopt osi s, thereby suppr essi ng the di f f er ent i at i on of ost eocl ast s, and the present i nvent i on i s compl et ed on the basis thereof.
[ Summary of I nvent i on]
[Techni cal Pr obl em]
Drug therapies for treating rheumatoid arthritis i ncl ude pri mary drugs such as nonsteroi dal anti -i nf I ammat ory drugs and st er oi d preparat i ons, whi ch are a ki nd of hormone, and secondary drugs that suppress rheumatoid arthritis itself by affecting the immune system of the body, and are usual 1 y used for a long period of time, thereby requiring attention to side effects. In particular, steroid preparations may be abused because good effects are exhi bi t ed i mmedi at el y, but pr obl ems of si de effects are ser i ous. The long- t erm use thereof may be a r i sk factor for ost eoporosi s.
Accor di ngl y, the present i nvent ors have made efforts to devel op a new t her apeut i c agent havi ng an effective therapeutic effect while minimizing the side effects of a therapeutic agent for arthritis, including rheumatoid arthritis, and/or osteoporosis. As a result, it is conf i rmed that stabilized gal ect i n 9 protein in which ami no aci ds of the C- t ermi nal domai n ( CCRD) of the two sugar chain recognition sites and of link peptides of the existing wild-type gal ect i n 9 are del et ed and substituted have an effect of suppr essi ng Thl and Th17 differentiation and i nduci ng f i brobl ast - I i ke synovi ocyt es ( FLS) apopt osi s, thereby suppr essi ng the di f f er ent i at i on of ost eocl ast s, and the present i nvent i on i s compl et ed on the basis thereof.
[ Summary of I nvent i on]
[Techni cal Pr obl em]
4 An obj ect of the present invention is to provide a recombi nant stabilized gal ect i n 9 prot ei n and a pharmaceutical composition for prevention or treatment of a bone di sease i ncl udi ng the same.
[Solution to Pr obl em]
In or der to achi eve the obj ect of the present i nvent i on, the present i nvent i on provi des a recombi nant stabilized gal ect i n 9 protein havi ng the ami no acid sequence represented by SEQ ID NO: 1.
I n addi ti on, the present i nvent i on provi des a recombi nant vector i ncl udi ng a gene that encodes the pr ot ei n.
I n addi ti on, the present i nvent i on provi des a t r ansf or mant i nt o whi ch the recombi nant vector i s i nser t ed.
I n addi ti on, the present i nvent i on provi des a pharmaceutical composition for prevention or treatment of a bone di sease, i ncl udi ng a recombi nant st abi I i zed gal ect i n 9 prot ei n havi ng an ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eot i de that encodes the same, as an active i ngredi ent; a met hod for treating a bone disease, including admi ni st er i ng, to an i ndi vi dual , a pharmaceut i cal I y effective amount of a recombi nant stabilized gal ect i n 9 prot ei n havi ng the ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eot i de encodi ng the same; a pharmaceut i cal composi ti on i ncl udi ng a recombi nant stabilized gal ect i n 9 pr ot ei n havi ng an ami no aci d sequence represented by SEQ
ID NO: 1 or a pol ynucl eot i de that encodes the same for use i n the prevent i on or treatment of a bone di sease; and the use of a recombi nant st abi I i zed gal ect i n 9 prot ei n havi ng an ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eot i de that encodes the same for the pr epar at i on of a phar maceut i cal composi ti on for pr event i on or treatment of a bone di sease.
Al so, the present i nvent i on pr ovi des a health f unct i onal food for pr event i on or i mpr ovement of a bone di sease, i ncl udi ng a recombi nant stabilized gal ect i n 9 pr ot ei n havi ng the ami no aci d sequence represented by SEQ
ID NO: 1, as an active ingredient; a met hod for pr event i ng or i mprovi ng a bone di sease, i ncl udi ng admi ni st er i ng, to an individual, a pharmaceutically effective amount of a recombinant stabilized gal ect i n 9 pr ot ei n havi ng the ami no aci d sequence represented by SEQ
ID NO: 1 or a pol ynucl eot i de that encodes the same; a health f unct i onal food i ncl udi ng a recombi nant stabilized gal ect i n 9 prot ei n havi ng an ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eot i de that encodes the same for use in prevention or improvement of a bone di sease; and the use of a recombi nant st abi I i zed gal ect i n 9 prot ei n havi ng an ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eot i de that encodes the same for the preparation of a health f unct i onal food for pr event i ng or i mpr ovi ng a bone di sease.
[Advantageous Effects of I nvent i on]
The recombi nant stabilized gal ect i n 9 protein of the present i nvent i on exhi bits Th1 and Th17 cell differentiation inhibitory effects, f i br obl ast - I i ke synovi ocyt es ( FLS) apopt osi s effects and ost eocl ast differentiation inhibitory effects, and thus can be usefully used as an active ingredient in a composition for pr event i on or treatment of a bone di sease i ncl udi ng rheumatoid arthritis.
[Brief Descr i pt i on of Dr awi ngs]
FIG. 1 is a figure showing a binding degree of a r ecombi nant stabilized gal ect i n 9 prot ei n ( sGal -9) according to the present invention to Ti m- 3.
FIG. 2 is a figure showing Thl and Th17 cell differentiation inhibitory effects of sGal -9 according to the present i nvent i on.
FIG. 3 is a figure showing f i brobl ast- I i ke synovi ocyt es ( FLS) apopt osi s effects of sGal - 9 according to the present i nvent i on.
Fl G. 4 is a fi gure showi ng osteocl ast differentiation inhibitory effects of sGal -9 according to the present i nvent i on.
FIG. 5 is a figure showing the bone resorption i nhi bi tory effect of sGal -9 accordi ng to the present i nvent i on.
[ Descr i pt i on of Embodi ment s]
Her ei naf t er , embodi ments of the present i nvent i on will be described in detail so that those of ordinary skill in the art to which the present invention pertains can easi I y carry out the present i nvent i on. The embodi ment s of the present i nvent i on are pr ovi ded i n or der to more completely explain the present invention to those of or di nary ski II in the art to whi ch the present i nvent i on pert ai ns. Accor di ngl y, the embodi ment of the present invention may be modified in van i ous other forms, and the scope of the present invention is not limited to the embodi ment s descr i bed bel ow.
Throughout the specification of the present invention, if it is said that any part " i ncl udes" any component, t hi s means that other components may be further i ncl uded, rat her than excl udi ng the other components, unl ess otherwi se stated.
The present i nventi on provi des a recombi nant stabilized gal ecti n 9 protein having an ami no acid sequence represented by SEQ ID NO: 1.
I n the present i nventi on, the recombi nant stabi I i zed gal ecti n 9 protein has a more stable molecular structure to a protease while maintaining the sugar chain recogni ti on activity of wild type Gal ecti n-9.
Speci f i cal I y, the recombi nant stabilized gal ecti n 9 protei n is a recombi nant protei n prepared by modi fyi ng a I i nk r egi on connect i ng two carbohydrate recogni ti on domains ( CRDs) of wild-type gal ecti n 9 having the structure of NCRD- I i nker-CCRD and a C-termi nal carbohydrate recogni ti on domai n ( CCRD) . More speci f i cal I y, the recombi nant stabilized gal ecti n 9 protein is obtained by deleting al I peptides of the I i nker regi on, del et i ng the ami no aci d sequence at positions 1 to 10 ( SEQ ID NO: 3) in CCRD ( SEQ ID NO: 2), and substituting Al a ( Al ani ne; A) at posi ti on 13 with Pro ( Prol i ne; P), may i ncl ude the ami no aci d sequence represented by SEQ I D NO: 1, may i ncl ude an ami no aci d sequence havi ng sequence homol ogy of 75% or more, preferably 80% or more, more preferably 90% or more with the ami no aci d sequence represented by SEQ I D NO: 1, and may further i ncl ude t ar get i ng sequences, tags, I abel ed resi dues, and an ami no aci d sequence prepared for a specific purpose to increase half-life or peptide stability.
I n addi ti on, the recombi nant protei n of the present i nventi on can be obtai ned by van i ous met hods wel I known in the art. As an example, the recombi nant protein of the present i nventi on may be prepared usi ng pol ynucl eoti de recombi nati on and protei n expressi on systems, may be synthesi zed i n vitro through chemi cal synthesi s such as pepti de synthesi s, or may be prepared by cell -free protei n synt hesi s.
As used herein, the term "pol ynucl eoti de" refers to a polymer to which nucleotides are bound, and serves to transmit genetic information. For the purposes of the present i nventi on, pol ynucl eoti de encodes the recombi nant protei n of SEQ I D NO: 1 and may i ncl ude sequence havi ng sequence homol ogy of 75% or more, pref erabl y 85% or more, more pref erabl y 90% or more, and most pref erabl y 95% or more with the pol ynucl eoti de sequence for encodi ng the recombi nant protei n.
As used herein, the term "homology" is intended to indicate a degree of similarity to a wild type amino acid sequence or a pol ynucl eoti de sequence, the comparison of such homol ogy can be performed usi ng a compari son program well known in the art, and homol ogy between two or more sequences can be cal cul at ed as a percentage (A).
I n addi ti on, the present i nventi on provi des a recombi nant vector i ncl udi ng a gene that encodes the protei n.
I n addi ti on, the present i nventi on provi des a transf ormant i nto whi ch the recombi nant vector i s i nsert ed.
As used herein, the term "vector" refers to a pl asmi d, a virus or other medium well-known in the art i nto whi eh a gene or a base sequence can be i nserted or i ntroduced, speci f i cal I y may be I i near DNA, pl asmi d DNA, a recombi nant non-vi ral vector, a recombi nant vi ral vector, or an i nduci bl e gene expressi on vector system, and the recombi nant vi ral vector may be a retrovi rus, an adenovi r us, an adeno-associ ated vi r us, a hel per-dependent adenovi r us, a herpes si mpl ex vi r us, a I enti vi ral vector or a vacci ni a virus, but the vector is not limited thereto. The base sequence accordi ng to the present i nventi on may be operably I i nked to an expressi on control sequence, and the operably linked base sequence may be i ncl uded i n one expressi on vector i ncl udi ng a sel ecti on marker and a replication origin together. Those that are "operably I i nked" can be a gene and an expressi on control sequence that are I i nked i n a manner that enabl es gene expressi on when an appropri ate mol ecul e i s bound to the expressi on control sequence. The term "expressi on control sequence" refers to a DNA sequence that control s the expressi on of an operabl y I i nked base sequence i n a speci f i c host cel I . Such control sequences i ncl ude promoters for effecting t r anscr i pt i on, any operator sequences for regul at i ng transcri pti on, sequences for encodi ng sui tabl e mRNA ri bosome bi ndi ng sites, and sequences for regul at i ng the t ermi nat i on of t ranscr i pt i on and translation.
As used her ei n, the term "transf ormati on" refers to a change i n genet i c properti es of an organi sm by a DNA
given from the outsi de, that i s, a phenomenon i n whi ch when a DNA, whi ch i s a type of nucl ei c aci d extracted from a cell of a certain lineage of an organism, is introduced into a living cell of another lineage, the DNA
enters the cel I and changes i n hereditary char act er i sti cs. The cel I may be a prokaryoti c cel I or a eukaryoti c cell, but is not limited thereto.
I n the present i nventi on, the gene that encodes the recombi nant protei n can be i ntroduced i nto cel I s after preparing a primer capable of specifically recognizing the gene from a known sequence as descri bed above, ampl i fyi ng the gene through a pol ymerase chai n react i on ( PCR) by using the pri mer, and introducing the amplified gene i nto the vector as descri bed above. The i ntroducti on methods are known, and examples thereof i ncl ude I i posome medi at ed transf ecti on, a cal ci um phosphate method, DEAF-dext ran medi at ed t r ansf ect i on, positively charged lipid medi at ed t ransf ect i on, el ect r opor at i on, t ransduct i on usi ng a phage system, and i nf ecti on method usi ng a vi r us.
However, the introduction met hods are not limited thereto.
I n addi ti on, the present i nventi on provi des a pharmaceutical composition for prevention or treatment of a bone di sease, i ncl udi ng the recombi nant stabi I i zed gal ecti n 9 protei n havi ng the ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eoti de that encodes the same, as an active i ngredi ent.
As used herein, the term "pr event i on" refers to any act i on that suppresses or del ays an onset of a di sease by admi ni strati on of a composi ti on.
As used herein, the term "treatment" refers to any act i on i n whi ch symptoms of a di sease are i mproved or benef i ci ally changed by admi ni strati on of the composi ti on.
I n the present i nvent i on, the recombi nant stabi I i zed gal ecti n 9 protein may exhibit one or more of the f ol I owi ng properti es, thereby havi ng an effect of pr event i ng or treati ng a bone di sease:
i ) increase in a degree of binding Ti m- 3 protein;
i i ) inhibition of Th1 and Th17 cell differentiation;
iii) inhibition of T helper cells;
iv) apoptosi s of f i brobl ast - I i ke synovi ocyt es ( FLS);
and v) i nhi bi ti on of osteocl ast di f f erent i at i on and bone r esor pt i on.
Speci f i cal I y, the recombi nant stabilized gal ecti n 9 protei n can i nhi bit hel per T cell s such as Th1 and Th17 through an apoptosi s i nducti on mechani sm by bi ndi ng to Ti m-3 protei n expressed on T cel Is.
I n part i cul ar, the synovi ocyt es are one of the important pat hol ogi es of rheumatoid arthritis together with inflammatory cytoki nes, and abnormal prol i f erat i on and act i vat i on of synovi ocyt es becomes a problem as the di sease progresses, and thus inflammation and destruction of joints may conti nuousl y occur.
In addition, the recombinant stabilized gal ect i n 9 protei n can cause apoptosi s of f i brobl ast - I i ke synovi ocyt es that destroys the matrix of articular cartilage by secreting matrix met hal I oprotei nase ( MMP) and cat hepsi n.
In addition, the recombinant stabilized gal ect i n 9 protein can inhibit osteocl ast differentiation and bone resorption. In particular, the ost eocl ast s are multi nucl eat ed cel 1 s that destroy and absorb bone ti ssues that are unnecessary i n bone growth, and if the bal ance with osteobl asts that are i nvol ved i n bone gener at i on and r egener at i on i s di sr upt ed, osteoporosi s that causes bone loss may occur. In addition, the bone resorption is a phenomenon in which bone is resorbed by the activity of bone- dest royi ng cel I s, and bi sphosphonat es that are general I y known osteoporosi s drugs i nhi bit the bone resorption to pr event the exacerbation of osteoporosis.
I n the present i nventi on, the bone di seases may be arthritis, osteoporosis, per i pr ost het i c ost eol ysi s, osteol ysi s caused by f at i gue debri s of i mpl ants, Paget' s di sease, osteomal aci a, ri ckets, osteopeni a, cal ci um dysregul at i on, met ast at i c bone cancer, i nf I ammatory bone I osses, secondary bone I osses by endocri ne di sease or drugs, or per i odont al di seases accompani ed by destructi on of al veol ar bone, but are not 1 i mi ted thereto.
In addition, the arthritis may be ost eoart hr i t i s, degenerative arthritis, rheumatoid arthritis, di ssoci at i ve osteochondri ti s, j oi nt I i gament damages, meni scus damages, j oi nt mi sal i gnment, avascul ar necrosi s or juvenile idiopathic arthritis, but is not limited thereto.
I n a speci f i c embodi ment of the present i nvent i on, the present i nvent ors I ysed and pun i f i ed E. col i cell s that i nduce the expressi on of the recombi nant stabilized gal ecti n 9 protei n havi ng the ami no aci d sequence represented by SEQ ID NO: 1, and then prepared a recombi nant protei n by usi ng a chromatography method. I n addi ti on, it was conf i rmed that the recombi nant protei n exhibited Thl and Th17 cell differentiation inhibitory effects, f i brobl ast- I i ke- synovi ocyt es ( FLS) apopt osi s effects and ost eocl ast di f f erent i at i on i nhi bi t or y effects. Therefore, the recombi nant protei n and the pol ynucl eoti de t hat encodes the same can be usef ul I y used as an active i ngredi ent of a composi ti on for prevent i on and treatment of a bone di sease.
Meanwhi I e, the recombi nant protei n of the present invention or a pol ynucl eoti de that encodes the same may be del i ver ed by phar maceut i cal I y accept abl e car r i er s such as col I oi dal suspensi on, powder, sal i ne, I i pi ds, I i posomes, mi crospher es, or nanospheri cal part i cl es.
These can form a complex with a vehicle or be associated therewith and can be delivered in vivo by using a del i very system wel I - known i n the art such as I i pi ds, I i posomes, mi croparti cl es, gol d nanopart i cl es, pol ymers, condensat i on reagents, pol ysacchari des, pol yami no aci ds, dendri mers, saponi ns, adsorpti on enhanci ng substances or fatty acids.
I n addi ti on, exampl es of pharmaceut i cal I y accept abl e car r i er s i ncl ude I act ose, dextrose, sucrose, sor bi t ol , manni t ol , starch, acaci a gum, cal ci um phosphate, al gi nate, gelatin, calcium silicate, mi crocryst al I i ne cel I ul ose, pol yvi nyl pyrrol i done, cell ul ose, water, syrup, methyl cell ul ose, methyl hydroxybenzoat e, pr opyl hydroxybenzoate, t al c, magnesi urn st ear at e and mi ner al oi I , whi ch are commonl y used i n prepar at i ons, but the pharmaceuti cal I y accept abl e car r i ers are not I i mi ted thereto. Further, in addition to the above components, a I ubri cant, a wetti ng agent, a sweet eni ng agent, a fl avori ng agent, an emul si fyi ng agent, a suspendi ng agent, a preservati ve, and the I i ke may be addi ti onal I y i ncl uded.
Accor di ng to a desi red method, the pharmaceuti cal composi ti on of the present i nventi on may be admi ni stered orally or parent eral I y (for example, intramuscularly, i nt ravenousl y, i nt raper i t oneal I y, subcutaneously, i ntradermal I y, or locally applied), and a dosage thereof may vary dependi ng on states and wei ghts of pat i ents, the severity of a disease, drug forms, routes of the admi ni strati on and ti me, but may be appropri at el y selected by those skilled in the art.
The pharmaceuti cal composi ti on of the present i nventi on i s admi ni stered i n a pharmaceuti cal I y effective amount.
As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio, which is applicable to medical treatments, and the effective dose level can be determi ned accordi ng to a pat i ent' s di sease type, severity, drug activities, sensitivity to a drug, admi ni strati on ti me, admi ni strati on route, excr et i on rate, treatment durati on, factors i ncl udi ng concurrent drugs and other factors well-known in the medical field.
The pharmaceuti cal composition according to the present i nventi on may be admi ni stered as an i ndi vi dual t her apeut i c agent, may be used i n combi nati on with sur ger i es, hormone t her api es, drug t her api es and bi ol ogi cal response modi f i ers, may be admi ni stered si mul taneousl y, separately, or sequenti ally with the agents, and may be admi ni stered i n a si ngl e does or multi pl e doses. It is i mport ant to admi ni ster an amount capable of obtaining the maxi mum effect with a minimum amount without si de effects i n consi der at i on of al I of the above factors, whi ch can be easi I y determi ned by those skilled in the art.
Specifically, the effective amount of the pharmaceuti cal composi ti on of the present i nventi on may vary dependi ng on a pat i ent' s age, sex, condi ti ons, wei ght, absorpti on of the active ingredient into the body, i nacti vat i on rate, excreti on rate, di sease type, and drugs used i n combi nati on, and may be i ncreased or decreased accordi ng to the admi ni strati on route, the severity of obesity, the sex, the wei ght, the age, and the like.
In addition, the present invention provides a health f unct i onal food for pr event i on or i mpr ovement of a bone di sease, i ncl udi ng the recombi nant stabi I i zed gal ecti n 9 protei n havi ng the ami no aci d sequence represented by SEQ
ID NO: 1, as an active ingredient.
In the present invention, the contents relating to the recombi nant stabilized gal ecti n 9 protei n and bone diseases are the same as those described above, and specific descriptions thereof refer to the above content.
As used herein, the term "improvement" refers to any acti ons that at I east reduces parameters rel ated to the condi ti on bei ng treated, for exampl e, the severity of symptoms. I n t hi s case, the health f uncti onal food composi ti on may be used before or after the onset of the di sease i n order to prevent or i mprove a bone di sease, simultaneously with or separately from a drug for treatment.
Meanwhi I e, i n the present i nvent i on, it was conf i r med that the r ecombi nant stabilized gal ect i n 9 pr ot ei n havi ng the ami no aci d sequence represented by SEQ
ID NO: 1 exhibits Th1 and Th17 cell differentiation inhibitory effects, f i br obl ast - I i ke- synovi ocyt es ( FLS) apopt osi s effects and ost eocl ast differentiation i nhi bi t or y effects. Therefore, the r ecombi nant pr ot ei n can be useful I y used as an active i ngr edi ent of a health f unct i onal food for pr event i on or i mpr ovement of a bone di sease.
In the health functional food of the present invention, the active ingredient may be added to food as it is or used together with other food or food i ngr edi ent s, and may be appr opr i at el y used accor di ng to a met hod in the related art. The mixing amount of the active i ngr edi ent s may be appr opr i at el y det er mi ned dependi ng on the purpose of its use (for pr event i on or i mpr ovement ) . In general, in the preparation of food or beverage, the health functional food of the present i nvent i on may be added i n an amount of pr ef er abl y 15% by wei ght or I ess and preferably 10% by wei ght or I ess, with respect to the raw material. However, if the purpose is for health and hygiene or in the case of long-term intake for the purpose of health control , the amount may be equal to or I ess than the above range.
The health functional food of the present invention may i ncl ude other i ngr edi ent s as essential i ngr edi ent s without any particular limitation other than the cont ai ni ng of the above active i ngr edi ent s. For exampl e, as i n general beverages, van i ous fl avor i ng agents, natural carbohydrates, or the I i ke may be i ncl uded as addi ti onal i ngredi ents. Exampl es of the above natural carbohydrates i ncl ude monosacchari des such as gl ucose and fructose; di sacchari des such as maltose and sucrose; and pal ysacchari des, for exampl e, general sugars such as dextri n and cycl odextri n, and sugar al cohol s such as xyl i t ol , sor bi t ol , and eryt hr i t ol .
In addi ti on to those descr i bed above, as fl avor i ng agents, natural fl avor i ng agents ( t haumati n, stevi a extract (for example, rebaudi osi de A and gl ycyr r hi zi n)) and synt het ic fl avori ng agents ( sacchari n, aspartame, and the like) may be advantageously used. The ratio of the natural carbohydrate may be appropri at el y determi ned by the selection of those skilled in the art.
In addition to the above, the health functional food of the present i nventi on may i ncl ude van i ous nut r i ents, vitamins, minerals ( el ectrol yt es) , flavoring agents such as synt het i c and natural fl avori ng agents, col or i ng agents and t hi ckeners ( cheese, chocol ate, and the I i ke), pectic acid and salts thereof, al gi ni c acid and salts thereof, or gani c aci ds, protective col I oi dal t hi ckeners, pH adj usters, stabi I i zers, preservati yes, gl yceri n, al cohol s, carbonates used i n carbonated beverages, and the I i ke. These components may be used i ndependent I y or i n combi nati on, and the proporti on of these addi ti yes may al so be appropri at el y sel ected by those skilled in the art.
I n addi ti on, the present i nventi on provi des a method for pr event i ng or t r eat i ng a bone di sease, i ncl udi ng admi ni steri ng, to an individual, a pharmaceutically effective amount of the recombi nant stabi I i zed gal ecti n 9 protei n havi ng the ami no aci d sequence represented by SEQ
ID NO: 1 or a pol ynucl eoti de that encodes the same.
I n addi ti on, the present i nventi on provi des a method f or pr event i ng or i mpr ovi ng a bone disease, i ncl udi ng admi ni st er i ng, to an individual, a pharmaceutically effective amount of the recombi nant st abi I i zed gal ect i n 9 pr ot ei n havi ng the ami no aci d sequence represented by SEQ
ID NO: 1 or a pol ynucl eot i de that encodes the same.
The recombi nant st abi I i zed gal ect i n 9 pr ot ei n havi ng the ami no aci d sequence represented by SEQ I D NO: 1 of the present i nvent i on or a pol ynucl eot i de that encodes the same exhibits Ti m-3 protein binding synergistic effects, Thl and Th17 cell differentiation i nhi bi t or y effects, hel per T cell inhibitory effects, f i br obl ast -I i ke- synovi ocyt es ( FLS) apopt osi s effects, ost eocl ast differentiation inhibitory effects and bone resorption inhibitory effects, and thus can be usefully used to treat a bone di sease.
I n addi ti on, the present i nvent i on provi des a pharmaceutical composition used for prevention or treatment of a bone di sease, i ncl udi ng the recombi nant stabilized gal ect i n 9 protein havi ng the ami no acid sequence represented by SEQ ID NO: 1 or a pol ynucl eot i de that encodes the same.
In addition, the present invention provi des a health f unct i onal food used for pr event i on or improvement of a bone di sease, i ncl udi ng the recombi nant st abi I i zed gal ect i n 9 prot ei n havi ng the ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eot i de that encodes the same.
I n addi ti on, the present i nvent i on provi des the use of the recombi nant stabilized gal ect i n 9 protein havi ng the ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eot i de that encodes the same for the preparation of a pharmaceut i cal composi ti on for pr event i on or treatment of a bone di sease.
I n addi ti on, the present i nvent i on provi des the use of the recombi nant stabilized gal ect i n 9 protein having the ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eot i de that encodes the same for the preparation of a health functional food for prevention or improvement of a bone di sease.
Her ei naf t er, the present i nvent i on i s be descr i bed in more detail through preparation exampl es and examples.
However, the f ol I owi ng pr epar at i on exampl es and exampl es are i nt ended to hel p the under st andi ng of the present invention and are not intended to limit the scope of the present i nvent i on.
<Pr epar at i on Exampl e 1> Pr epar at i on of Recombi nant Stabilized Gal ect i n 9 Protein An expr essi on vector i ncl udi ng a gene that encodes the recombi nant stabilized gal ect i n 9 protein having the ami no aci d sequence of SEQ I D NO: 1 was prepared, and the expr essi on vector was i nt r oduced i nt o E. col i by a heat shock method. The recombi nant pr ot ei n was expressed by culturing E. col i in an LB medium including 50 pg/ ml of kanamyci n and addi ng ar abi nose when the absorbance at 600 nm reached 0.7 to induce the expression of the recombi nant prot ei n. Then, the cel I s i nduced to express the recombi nant protein were I ysed and filtered, and the target protei n was captured by usi ng a cat i on exchange method, an affinity column, and the like to obtain a highly purified recombi nant stabilized gal ect i n 9 protein in a high yield.
<Exampl e 1> Conf i r mat i on of Ti m-3 Protein Binding Ability of Recombi nant St abl e Gal ect i n 9 Pr ot ei n ( sGal -9) 100 pl of recombinant quman im-3 (R&D systems, Cat.
No. 10241-1I-050) at a concentration of 0.5 pg/ml was added to each well of a 96-well ELI SA plate (Nunc, Cat.
No. 44-2404-21), the reaction was carried out at room temperature for one night, and then the resultant was washed three times with DPBS (Dulbecco's phosphate-buffered saline; Wel gene, Cat. No. LB011-02) including 0.05% Tween 20 (Sigma Cat. No. P9416). After washing, 200 pl of DPBS including 1% bovine serum albumin (BSA) was added to each well, incubated at room temperature for one hour, and washed three times with a wash buffer. After washing three times, the recombinant stabilized galectin 9 protein (sGal-9) obtained in <Preparation Example 1>
was diluted to various concentrations (0, 5, 10 and 20 pg/ml) and added by 100 pl to each well, incubated at room temperature for two hours, and washed three times with a wash buffer.
For an experimental group, Gal-9 antibody (R&D
systems, Cat. No. 1015238) was diluted to 1:1000 by using 1% BSA/DP3S and added by 100 n1 to eacq well, incubated at room temperature for one hour, and washed three times with a wash buffer. As a control group, an anti-mouse HRP
antibody (lnvitrogen, Cat. No. 31430) was diluted to 1:1000 by using 1% BSA/DPBS and added by 100 pl to each well, incubated at room temperature for one hour, and washed three times with a wash buffer.
100 pl of a 1:1 mixture of substrate reagents (R&D
systems, Cat. No. DY999) A and B was added to each washed well and incubated at room temperature with the light blocked for 20 minutes. After the incubation, 50 pl of a stop solution (2 N H2SO4) was added to each well, and the optical density (0.D) value was measured and checked at 450 nm using a microplate reader.
As a result, as shown in FIG. 1, it was confirmed that the optical density increased as the concentration of sGal -9 i ncr eased, and thus it was conf i rmed that the binding degree of sGal - 9 and Ti m- 3 was excellent in a concent r at i on-dependent manner ( Fl G. 1) .
<Exampl e 2> Conf i r mat i on of T Hel per Cell Differentiation Inhibitory Effect of sGal - 9 Anti - CD3 ( eBi osci ence, Cat. No. 16-0031-82) at a concentration of 2 pg/m1 diluted with PBS (Welgene, Cat.
No. LB 001-02) was added to a 96-well plate by 100 p1 or to a 24-well plate by 500 u11 was reacted at 4 C for one ni ght, was washed once with PBS, and was used for cel 1 culture. After the resultant was dispensed into 96-well pl at es or 24- wel 1 pl at es coated with CD4, CD62L and T
cells and incubated at 37 C and 5% CO2 condi ti ons for four days, ant i -1 L-4, ant i - I L-2 and ant i -1 L-12 cyt oki nes were added to i nduce the di f f erent i at i on i nt o Th1 cell s and anti-IFN-y, anti-IL-4, anti-IL-2 and anti-IL-6 cytokines were added to i nduce di f f erent i at i on i nto Th17 cells.
After the addition of cytoki nes, sGal - 9 with a concentration of 1 lag/m1 was added and incubated at 37 C
and 5% CO2 condi ti ons for four days.
After the culture, IFN-y cells of ml cells and IL-17 cytoki nes of Th17 cells were measured by usi ng an ELI SA assay kit ( Mouse DuoSet, R&D Systems) according to the procedure of the manufacturer.
As a result, as shown in FIG. 2, it was confirmed that the di f f erent i at i on of Th1 and Th17 cell s whi ch are the helper T cells was inhibited as the concentration of sGal - 9 increased, and thus it was confirmed that the hel per T cell differentiation inhibitory effect of sGal -9 was excellent ( Fl G. 2) .
<Exampl e 3> Conf i r mat i on of Fi brobl ast- I i ke-synovi ocyt es ( FLS) Apoptosi s Effect of sGal - 9 200 pl of the FLS cells (cell passage: RA
_______________________________________________________________ FLS 2-92#P7) were di spensed i nto each well of a 48- wel I pl ate (SPL, Cat. No. 30048) at a cell density of 5 x 104 cells/ml, and after 24 hours, 100 pl of sGal-9 at various concentrations (2.5, 5, and 10 pg/ml) was added to each wel I . After 16 hours, the supernatant was col I ect ed and stored in a deep freezer.
Cell s for fl uorescence act i vat ed cell sort i ng ( FACS) were placed and washed in a 15 ml tube, 200 pl of FACS
buffer was added thereto, and the resultant was stored at 4 C for one hour and was stai ned with Annexi n V- Fl TC/ 7-AAD ( Bi ol egend, Cat. No. 640922) . The stai ned cells were filtered witq a 70 pm cell strainer (3D Falcon, Cat. No.
352350) into a tube for FACS.
As a result, as shown in FIG. 3, it was confirmed that the f i brobl ast - I i ke- synovi ocyt es apopt osi s effect i ncr eased as the concent r at i on of sGal -9 i ncr eased, and thus it was conf i rmed that the synovi al cell apoptosi s effect of sGal - 9 was excellent ( Fl G. 3) .
<Exampl e 4> Conf i r mat i on of Osteocl ast Di f f erent i at i on Inhibitory Effect of sGal - 9 1 ml of osteocl asts was passaged i n each wel I of a 12- wel I pl ate ( Corni ng, Cat. No. 3513) at a cell densi ty of 2.5 x 105 cel I s/ ml , and after 24 hours, sGal - 9 as an exper i mental group at van i ous concent r at i ons (0. 1, 0. 5, 1, 2, 4, and 10 ag/m1)and PBS as a control group were put into each well by 1 ml.
After compl et i on of di f f er ent i at i on, the stai ned osteocl asts of the experimental group and the control group were TRAP- stai ned by usi ng the TRAP St ai ni ng Kit ( K- ASSAY, Cat. No. KT-008) and counted by using an opt i cal mi croscope, and then multi nucl eat ed cel I s (TRAP( +) MNCs) i n whi ch three or more nucl ei were observed was determined to be differentiated into osteocl asts.
As a result, as shown in FIG. 4, it was confirmed that the number of stai ned osteocl asts decreased as the concent r at i on of sGal -9 i ncr eased, and thus it was conf i rmed that the osteocl ast differentiation inhibitory effect of sGal -9 was excellent ( Fl G. 4).
<Exampl e 5> Conf i r mat i on of Bone Resorpt i on I nhi bi tory Effect of sGal -9 1 ml of osteocl asts was passaged at a cel I densi ty of 2.5 x 105 cell s/ ml into each well of Osteo Assay Surf ace multi-well plate ( Corni ng, Cat. No. 3987) coated with i nor gani c three-di mensi onal crystal I i ne that resembled the bone structure in vi vo, and after 24 hours, sGal -9 as an experi mental group at van i ous concent r at i ons (0.1, 0.5, 1, 2, /, and 10 pg/m1)and PBS as a control group were put i nto each wel I by 1 ml . The experi mental group ( sGal -9), the control ( PBS), and cell culture medium ( MEM-al pha, gi bco, Cat. No. 12561-056) were repl aced daily unt i I di f f erent i at i on was compl et ed.
After completion of differentiation, cells were removed with 20% SOS (Bleaching solution), and the Osteo Assay Surf ace multi -wel I pl ate was observed under a mi croscope.
As a result, as shown in FIG. 5, it was confirmed that the bone resorpti on inhibitory effect of sGal -9 was excellent ( Fl G. 5).
I n concl usi on, through the results of <Exampl e 1> to <Exampl e 5>, it was confirmed that bone diseases could be prevented or treated by sGal -9 i n a concent rat i on-dependent manner, through the i ncrease i n the Ti m-3 protei n bi ndi ng ability, the i ncreases i n the Thl and Th17 cell di f f erent i at i on i nhi bi t or y effect and the synovi al cel I apoptosi s effect, and the i ncr eases i n the ost eocl ast di f f erent i at i on i nhi bi t or y effect and the bone r esor pt i on i nhi bi t or y effect.
[Solution to Pr obl em]
In or der to achi eve the obj ect of the present i nvent i on, the present i nvent i on provi des a recombi nant stabilized gal ect i n 9 protein havi ng the ami no acid sequence represented by SEQ ID NO: 1.
I n addi ti on, the present i nvent i on provi des a recombi nant vector i ncl udi ng a gene that encodes the pr ot ei n.
I n addi ti on, the present i nvent i on provi des a t r ansf or mant i nt o whi ch the recombi nant vector i s i nser t ed.
I n addi ti on, the present i nvent i on provi des a pharmaceutical composition for prevention or treatment of a bone di sease, i ncl udi ng a recombi nant st abi I i zed gal ect i n 9 prot ei n havi ng an ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eot i de that encodes the same, as an active i ngredi ent; a met hod for treating a bone disease, including admi ni st er i ng, to an i ndi vi dual , a pharmaceut i cal I y effective amount of a recombi nant stabilized gal ect i n 9 prot ei n havi ng the ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eot i de encodi ng the same; a pharmaceut i cal composi ti on i ncl udi ng a recombi nant stabilized gal ect i n 9 pr ot ei n havi ng an ami no aci d sequence represented by SEQ
ID NO: 1 or a pol ynucl eot i de that encodes the same for use i n the prevent i on or treatment of a bone di sease; and the use of a recombi nant st abi I i zed gal ect i n 9 prot ei n havi ng an ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eot i de that encodes the same for the pr epar at i on of a phar maceut i cal composi ti on for pr event i on or treatment of a bone di sease.
Al so, the present i nvent i on pr ovi des a health f unct i onal food for pr event i on or i mpr ovement of a bone di sease, i ncl udi ng a recombi nant stabilized gal ect i n 9 pr ot ei n havi ng the ami no aci d sequence represented by SEQ
ID NO: 1, as an active ingredient; a met hod for pr event i ng or i mprovi ng a bone di sease, i ncl udi ng admi ni st er i ng, to an individual, a pharmaceutically effective amount of a recombinant stabilized gal ect i n 9 pr ot ei n havi ng the ami no aci d sequence represented by SEQ
ID NO: 1 or a pol ynucl eot i de that encodes the same; a health f unct i onal food i ncl udi ng a recombi nant stabilized gal ect i n 9 prot ei n havi ng an ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eot i de that encodes the same for use in prevention or improvement of a bone di sease; and the use of a recombi nant st abi I i zed gal ect i n 9 prot ei n havi ng an ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eot i de that encodes the same for the preparation of a health f unct i onal food for pr event i ng or i mpr ovi ng a bone di sease.
[Advantageous Effects of I nvent i on]
The recombi nant stabilized gal ect i n 9 protein of the present i nvent i on exhi bits Th1 and Th17 cell differentiation inhibitory effects, f i br obl ast - I i ke synovi ocyt es ( FLS) apopt osi s effects and ost eocl ast differentiation inhibitory effects, and thus can be usefully used as an active ingredient in a composition for pr event i on or treatment of a bone di sease i ncl udi ng rheumatoid arthritis.
[Brief Descr i pt i on of Dr awi ngs]
FIG. 1 is a figure showing a binding degree of a r ecombi nant stabilized gal ect i n 9 prot ei n ( sGal -9) according to the present invention to Ti m- 3.
FIG. 2 is a figure showing Thl and Th17 cell differentiation inhibitory effects of sGal -9 according to the present i nvent i on.
FIG. 3 is a figure showing f i brobl ast- I i ke synovi ocyt es ( FLS) apopt osi s effects of sGal - 9 according to the present i nvent i on.
Fl G. 4 is a fi gure showi ng osteocl ast differentiation inhibitory effects of sGal -9 according to the present i nvent i on.
FIG. 5 is a figure showing the bone resorption i nhi bi tory effect of sGal -9 accordi ng to the present i nvent i on.
[ Descr i pt i on of Embodi ment s]
Her ei naf t er , embodi ments of the present i nvent i on will be described in detail so that those of ordinary skill in the art to which the present invention pertains can easi I y carry out the present i nvent i on. The embodi ment s of the present i nvent i on are pr ovi ded i n or der to more completely explain the present invention to those of or di nary ski II in the art to whi ch the present i nvent i on pert ai ns. Accor di ngl y, the embodi ment of the present invention may be modified in van i ous other forms, and the scope of the present invention is not limited to the embodi ment s descr i bed bel ow.
Throughout the specification of the present invention, if it is said that any part " i ncl udes" any component, t hi s means that other components may be further i ncl uded, rat her than excl udi ng the other components, unl ess otherwi se stated.
The present i nventi on provi des a recombi nant stabilized gal ecti n 9 protein having an ami no acid sequence represented by SEQ ID NO: 1.
I n the present i nventi on, the recombi nant stabi I i zed gal ecti n 9 protein has a more stable molecular structure to a protease while maintaining the sugar chain recogni ti on activity of wild type Gal ecti n-9.
Speci f i cal I y, the recombi nant stabilized gal ecti n 9 protei n is a recombi nant protei n prepared by modi fyi ng a I i nk r egi on connect i ng two carbohydrate recogni ti on domains ( CRDs) of wild-type gal ecti n 9 having the structure of NCRD- I i nker-CCRD and a C-termi nal carbohydrate recogni ti on domai n ( CCRD) . More speci f i cal I y, the recombi nant stabilized gal ecti n 9 protein is obtained by deleting al I peptides of the I i nker regi on, del et i ng the ami no aci d sequence at positions 1 to 10 ( SEQ ID NO: 3) in CCRD ( SEQ ID NO: 2), and substituting Al a ( Al ani ne; A) at posi ti on 13 with Pro ( Prol i ne; P), may i ncl ude the ami no aci d sequence represented by SEQ I D NO: 1, may i ncl ude an ami no aci d sequence havi ng sequence homol ogy of 75% or more, preferably 80% or more, more preferably 90% or more with the ami no aci d sequence represented by SEQ I D NO: 1, and may further i ncl ude t ar get i ng sequences, tags, I abel ed resi dues, and an ami no aci d sequence prepared for a specific purpose to increase half-life or peptide stability.
I n addi ti on, the recombi nant protei n of the present i nventi on can be obtai ned by van i ous met hods wel I known in the art. As an example, the recombi nant protein of the present i nventi on may be prepared usi ng pol ynucl eoti de recombi nati on and protei n expressi on systems, may be synthesi zed i n vitro through chemi cal synthesi s such as pepti de synthesi s, or may be prepared by cell -free protei n synt hesi s.
As used herein, the term "pol ynucl eoti de" refers to a polymer to which nucleotides are bound, and serves to transmit genetic information. For the purposes of the present i nventi on, pol ynucl eoti de encodes the recombi nant protei n of SEQ I D NO: 1 and may i ncl ude sequence havi ng sequence homol ogy of 75% or more, pref erabl y 85% or more, more pref erabl y 90% or more, and most pref erabl y 95% or more with the pol ynucl eoti de sequence for encodi ng the recombi nant protei n.
As used herein, the term "homology" is intended to indicate a degree of similarity to a wild type amino acid sequence or a pol ynucl eoti de sequence, the comparison of such homol ogy can be performed usi ng a compari son program well known in the art, and homol ogy between two or more sequences can be cal cul at ed as a percentage (A).
I n addi ti on, the present i nventi on provi des a recombi nant vector i ncl udi ng a gene that encodes the protei n.
I n addi ti on, the present i nventi on provi des a transf ormant i nto whi ch the recombi nant vector i s i nsert ed.
As used herein, the term "vector" refers to a pl asmi d, a virus or other medium well-known in the art i nto whi eh a gene or a base sequence can be i nserted or i ntroduced, speci f i cal I y may be I i near DNA, pl asmi d DNA, a recombi nant non-vi ral vector, a recombi nant vi ral vector, or an i nduci bl e gene expressi on vector system, and the recombi nant vi ral vector may be a retrovi rus, an adenovi r us, an adeno-associ ated vi r us, a hel per-dependent adenovi r us, a herpes si mpl ex vi r us, a I enti vi ral vector or a vacci ni a virus, but the vector is not limited thereto. The base sequence accordi ng to the present i nventi on may be operably I i nked to an expressi on control sequence, and the operably linked base sequence may be i ncl uded i n one expressi on vector i ncl udi ng a sel ecti on marker and a replication origin together. Those that are "operably I i nked" can be a gene and an expressi on control sequence that are I i nked i n a manner that enabl es gene expressi on when an appropri ate mol ecul e i s bound to the expressi on control sequence. The term "expressi on control sequence" refers to a DNA sequence that control s the expressi on of an operabl y I i nked base sequence i n a speci f i c host cel I . Such control sequences i ncl ude promoters for effecting t r anscr i pt i on, any operator sequences for regul at i ng transcri pti on, sequences for encodi ng sui tabl e mRNA ri bosome bi ndi ng sites, and sequences for regul at i ng the t ermi nat i on of t ranscr i pt i on and translation.
As used her ei n, the term "transf ormati on" refers to a change i n genet i c properti es of an organi sm by a DNA
given from the outsi de, that i s, a phenomenon i n whi ch when a DNA, whi ch i s a type of nucl ei c aci d extracted from a cell of a certain lineage of an organism, is introduced into a living cell of another lineage, the DNA
enters the cel I and changes i n hereditary char act er i sti cs. The cel I may be a prokaryoti c cel I or a eukaryoti c cell, but is not limited thereto.
I n the present i nventi on, the gene that encodes the recombi nant protei n can be i ntroduced i nto cel I s after preparing a primer capable of specifically recognizing the gene from a known sequence as descri bed above, ampl i fyi ng the gene through a pol ymerase chai n react i on ( PCR) by using the pri mer, and introducing the amplified gene i nto the vector as descri bed above. The i ntroducti on methods are known, and examples thereof i ncl ude I i posome medi at ed transf ecti on, a cal ci um phosphate method, DEAF-dext ran medi at ed t r ansf ect i on, positively charged lipid medi at ed t ransf ect i on, el ect r opor at i on, t ransduct i on usi ng a phage system, and i nf ecti on method usi ng a vi r us.
However, the introduction met hods are not limited thereto.
I n addi ti on, the present i nventi on provi des a pharmaceutical composition for prevention or treatment of a bone di sease, i ncl udi ng the recombi nant stabi I i zed gal ecti n 9 protei n havi ng the ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eoti de that encodes the same, as an active i ngredi ent.
As used herein, the term "pr event i on" refers to any act i on that suppresses or del ays an onset of a di sease by admi ni strati on of a composi ti on.
As used herein, the term "treatment" refers to any act i on i n whi ch symptoms of a di sease are i mproved or benef i ci ally changed by admi ni strati on of the composi ti on.
I n the present i nvent i on, the recombi nant stabi I i zed gal ecti n 9 protein may exhibit one or more of the f ol I owi ng properti es, thereby havi ng an effect of pr event i ng or treati ng a bone di sease:
i ) increase in a degree of binding Ti m- 3 protein;
i i ) inhibition of Th1 and Th17 cell differentiation;
iii) inhibition of T helper cells;
iv) apoptosi s of f i brobl ast - I i ke synovi ocyt es ( FLS);
and v) i nhi bi ti on of osteocl ast di f f erent i at i on and bone r esor pt i on.
Speci f i cal I y, the recombi nant stabilized gal ecti n 9 protei n can i nhi bit hel per T cell s such as Th1 and Th17 through an apoptosi s i nducti on mechani sm by bi ndi ng to Ti m-3 protei n expressed on T cel Is.
I n part i cul ar, the synovi ocyt es are one of the important pat hol ogi es of rheumatoid arthritis together with inflammatory cytoki nes, and abnormal prol i f erat i on and act i vat i on of synovi ocyt es becomes a problem as the di sease progresses, and thus inflammation and destruction of joints may conti nuousl y occur.
In addition, the recombinant stabilized gal ect i n 9 protei n can cause apoptosi s of f i brobl ast - I i ke synovi ocyt es that destroys the matrix of articular cartilage by secreting matrix met hal I oprotei nase ( MMP) and cat hepsi n.
In addition, the recombinant stabilized gal ect i n 9 protein can inhibit osteocl ast differentiation and bone resorption. In particular, the ost eocl ast s are multi nucl eat ed cel 1 s that destroy and absorb bone ti ssues that are unnecessary i n bone growth, and if the bal ance with osteobl asts that are i nvol ved i n bone gener at i on and r egener at i on i s di sr upt ed, osteoporosi s that causes bone loss may occur. In addition, the bone resorption is a phenomenon in which bone is resorbed by the activity of bone- dest royi ng cel I s, and bi sphosphonat es that are general I y known osteoporosi s drugs i nhi bit the bone resorption to pr event the exacerbation of osteoporosis.
I n the present i nventi on, the bone di seases may be arthritis, osteoporosis, per i pr ost het i c ost eol ysi s, osteol ysi s caused by f at i gue debri s of i mpl ants, Paget' s di sease, osteomal aci a, ri ckets, osteopeni a, cal ci um dysregul at i on, met ast at i c bone cancer, i nf I ammatory bone I osses, secondary bone I osses by endocri ne di sease or drugs, or per i odont al di seases accompani ed by destructi on of al veol ar bone, but are not 1 i mi ted thereto.
In addition, the arthritis may be ost eoart hr i t i s, degenerative arthritis, rheumatoid arthritis, di ssoci at i ve osteochondri ti s, j oi nt I i gament damages, meni scus damages, j oi nt mi sal i gnment, avascul ar necrosi s or juvenile idiopathic arthritis, but is not limited thereto.
I n a speci f i c embodi ment of the present i nvent i on, the present i nvent ors I ysed and pun i f i ed E. col i cell s that i nduce the expressi on of the recombi nant stabilized gal ecti n 9 protei n havi ng the ami no aci d sequence represented by SEQ ID NO: 1, and then prepared a recombi nant protei n by usi ng a chromatography method. I n addi ti on, it was conf i rmed that the recombi nant protei n exhibited Thl and Th17 cell differentiation inhibitory effects, f i brobl ast- I i ke- synovi ocyt es ( FLS) apopt osi s effects and ost eocl ast di f f erent i at i on i nhi bi t or y effects. Therefore, the recombi nant protei n and the pol ynucl eoti de t hat encodes the same can be usef ul I y used as an active i ngredi ent of a composi ti on for prevent i on and treatment of a bone di sease.
Meanwhi I e, the recombi nant protei n of the present invention or a pol ynucl eoti de that encodes the same may be del i ver ed by phar maceut i cal I y accept abl e car r i er s such as col I oi dal suspensi on, powder, sal i ne, I i pi ds, I i posomes, mi crospher es, or nanospheri cal part i cl es.
These can form a complex with a vehicle or be associated therewith and can be delivered in vivo by using a del i very system wel I - known i n the art such as I i pi ds, I i posomes, mi croparti cl es, gol d nanopart i cl es, pol ymers, condensat i on reagents, pol ysacchari des, pol yami no aci ds, dendri mers, saponi ns, adsorpti on enhanci ng substances or fatty acids.
I n addi ti on, exampl es of pharmaceut i cal I y accept abl e car r i er s i ncl ude I act ose, dextrose, sucrose, sor bi t ol , manni t ol , starch, acaci a gum, cal ci um phosphate, al gi nate, gelatin, calcium silicate, mi crocryst al I i ne cel I ul ose, pol yvi nyl pyrrol i done, cell ul ose, water, syrup, methyl cell ul ose, methyl hydroxybenzoat e, pr opyl hydroxybenzoate, t al c, magnesi urn st ear at e and mi ner al oi I , whi ch are commonl y used i n prepar at i ons, but the pharmaceuti cal I y accept abl e car r i ers are not I i mi ted thereto. Further, in addition to the above components, a I ubri cant, a wetti ng agent, a sweet eni ng agent, a fl avori ng agent, an emul si fyi ng agent, a suspendi ng agent, a preservati ve, and the I i ke may be addi ti onal I y i ncl uded.
Accor di ng to a desi red method, the pharmaceuti cal composi ti on of the present i nventi on may be admi ni stered orally or parent eral I y (for example, intramuscularly, i nt ravenousl y, i nt raper i t oneal I y, subcutaneously, i ntradermal I y, or locally applied), and a dosage thereof may vary dependi ng on states and wei ghts of pat i ents, the severity of a disease, drug forms, routes of the admi ni strati on and ti me, but may be appropri at el y selected by those skilled in the art.
The pharmaceuti cal composi ti on of the present i nventi on i s admi ni stered i n a pharmaceuti cal I y effective amount.
As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio, which is applicable to medical treatments, and the effective dose level can be determi ned accordi ng to a pat i ent' s di sease type, severity, drug activities, sensitivity to a drug, admi ni strati on ti me, admi ni strati on route, excr et i on rate, treatment durati on, factors i ncl udi ng concurrent drugs and other factors well-known in the medical field.
The pharmaceuti cal composition according to the present i nventi on may be admi ni stered as an i ndi vi dual t her apeut i c agent, may be used i n combi nati on with sur ger i es, hormone t her api es, drug t her api es and bi ol ogi cal response modi f i ers, may be admi ni stered si mul taneousl y, separately, or sequenti ally with the agents, and may be admi ni stered i n a si ngl e does or multi pl e doses. It is i mport ant to admi ni ster an amount capable of obtaining the maxi mum effect with a minimum amount without si de effects i n consi der at i on of al I of the above factors, whi ch can be easi I y determi ned by those skilled in the art.
Specifically, the effective amount of the pharmaceuti cal composi ti on of the present i nventi on may vary dependi ng on a pat i ent' s age, sex, condi ti ons, wei ght, absorpti on of the active ingredient into the body, i nacti vat i on rate, excreti on rate, di sease type, and drugs used i n combi nati on, and may be i ncreased or decreased accordi ng to the admi ni strati on route, the severity of obesity, the sex, the wei ght, the age, and the like.
In addition, the present invention provides a health f unct i onal food for pr event i on or i mpr ovement of a bone di sease, i ncl udi ng the recombi nant stabi I i zed gal ecti n 9 protei n havi ng the ami no aci d sequence represented by SEQ
ID NO: 1, as an active ingredient.
In the present invention, the contents relating to the recombi nant stabilized gal ecti n 9 protei n and bone diseases are the same as those described above, and specific descriptions thereof refer to the above content.
As used herein, the term "improvement" refers to any acti ons that at I east reduces parameters rel ated to the condi ti on bei ng treated, for exampl e, the severity of symptoms. I n t hi s case, the health f uncti onal food composi ti on may be used before or after the onset of the di sease i n order to prevent or i mprove a bone di sease, simultaneously with or separately from a drug for treatment.
Meanwhi I e, i n the present i nvent i on, it was conf i r med that the r ecombi nant stabilized gal ect i n 9 pr ot ei n havi ng the ami no aci d sequence represented by SEQ
ID NO: 1 exhibits Th1 and Th17 cell differentiation inhibitory effects, f i br obl ast - I i ke- synovi ocyt es ( FLS) apopt osi s effects and ost eocl ast differentiation i nhi bi t or y effects. Therefore, the r ecombi nant pr ot ei n can be useful I y used as an active i ngr edi ent of a health f unct i onal food for pr event i on or i mpr ovement of a bone di sease.
In the health functional food of the present invention, the active ingredient may be added to food as it is or used together with other food or food i ngr edi ent s, and may be appr opr i at el y used accor di ng to a met hod in the related art. The mixing amount of the active i ngr edi ent s may be appr opr i at el y det er mi ned dependi ng on the purpose of its use (for pr event i on or i mpr ovement ) . In general, in the preparation of food or beverage, the health functional food of the present i nvent i on may be added i n an amount of pr ef er abl y 15% by wei ght or I ess and preferably 10% by wei ght or I ess, with respect to the raw material. However, if the purpose is for health and hygiene or in the case of long-term intake for the purpose of health control , the amount may be equal to or I ess than the above range.
The health functional food of the present invention may i ncl ude other i ngr edi ent s as essential i ngr edi ent s without any particular limitation other than the cont ai ni ng of the above active i ngr edi ent s. For exampl e, as i n general beverages, van i ous fl avor i ng agents, natural carbohydrates, or the I i ke may be i ncl uded as addi ti onal i ngredi ents. Exampl es of the above natural carbohydrates i ncl ude monosacchari des such as gl ucose and fructose; di sacchari des such as maltose and sucrose; and pal ysacchari des, for exampl e, general sugars such as dextri n and cycl odextri n, and sugar al cohol s such as xyl i t ol , sor bi t ol , and eryt hr i t ol .
In addi ti on to those descr i bed above, as fl avor i ng agents, natural fl avor i ng agents ( t haumati n, stevi a extract (for example, rebaudi osi de A and gl ycyr r hi zi n)) and synt het ic fl avori ng agents ( sacchari n, aspartame, and the like) may be advantageously used. The ratio of the natural carbohydrate may be appropri at el y determi ned by the selection of those skilled in the art.
In addition to the above, the health functional food of the present i nventi on may i ncl ude van i ous nut r i ents, vitamins, minerals ( el ectrol yt es) , flavoring agents such as synt het i c and natural fl avori ng agents, col or i ng agents and t hi ckeners ( cheese, chocol ate, and the I i ke), pectic acid and salts thereof, al gi ni c acid and salts thereof, or gani c aci ds, protective col I oi dal t hi ckeners, pH adj usters, stabi I i zers, preservati yes, gl yceri n, al cohol s, carbonates used i n carbonated beverages, and the I i ke. These components may be used i ndependent I y or i n combi nati on, and the proporti on of these addi ti yes may al so be appropri at el y sel ected by those skilled in the art.
I n addi ti on, the present i nventi on provi des a method for pr event i ng or t r eat i ng a bone di sease, i ncl udi ng admi ni steri ng, to an individual, a pharmaceutically effective amount of the recombi nant stabi I i zed gal ecti n 9 protei n havi ng the ami no aci d sequence represented by SEQ
ID NO: 1 or a pol ynucl eoti de that encodes the same.
I n addi ti on, the present i nventi on provi des a method f or pr event i ng or i mpr ovi ng a bone disease, i ncl udi ng admi ni st er i ng, to an individual, a pharmaceutically effective amount of the recombi nant st abi I i zed gal ect i n 9 pr ot ei n havi ng the ami no aci d sequence represented by SEQ
ID NO: 1 or a pol ynucl eot i de that encodes the same.
The recombi nant st abi I i zed gal ect i n 9 pr ot ei n havi ng the ami no aci d sequence represented by SEQ I D NO: 1 of the present i nvent i on or a pol ynucl eot i de that encodes the same exhibits Ti m-3 protein binding synergistic effects, Thl and Th17 cell differentiation i nhi bi t or y effects, hel per T cell inhibitory effects, f i br obl ast -I i ke- synovi ocyt es ( FLS) apopt osi s effects, ost eocl ast differentiation inhibitory effects and bone resorption inhibitory effects, and thus can be usefully used to treat a bone di sease.
I n addi ti on, the present i nvent i on provi des a pharmaceutical composition used for prevention or treatment of a bone di sease, i ncl udi ng the recombi nant stabilized gal ect i n 9 protein havi ng the ami no acid sequence represented by SEQ ID NO: 1 or a pol ynucl eot i de that encodes the same.
In addition, the present invention provi des a health f unct i onal food used for pr event i on or improvement of a bone di sease, i ncl udi ng the recombi nant st abi I i zed gal ect i n 9 prot ei n havi ng the ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eot i de that encodes the same.
I n addi ti on, the present i nvent i on provi des the use of the recombi nant stabilized gal ect i n 9 protein havi ng the ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eot i de that encodes the same for the preparation of a pharmaceut i cal composi ti on for pr event i on or treatment of a bone di sease.
I n addi ti on, the present i nvent i on provi des the use of the recombi nant stabilized gal ect i n 9 protein having the ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eot i de that encodes the same for the preparation of a health functional food for prevention or improvement of a bone di sease.
Her ei naf t er, the present i nvent i on i s be descr i bed in more detail through preparation exampl es and examples.
However, the f ol I owi ng pr epar at i on exampl es and exampl es are i nt ended to hel p the under st andi ng of the present invention and are not intended to limit the scope of the present i nvent i on.
<Pr epar at i on Exampl e 1> Pr epar at i on of Recombi nant Stabilized Gal ect i n 9 Protein An expr essi on vector i ncl udi ng a gene that encodes the recombi nant stabilized gal ect i n 9 protein having the ami no aci d sequence of SEQ I D NO: 1 was prepared, and the expr essi on vector was i nt r oduced i nt o E. col i by a heat shock method. The recombi nant pr ot ei n was expressed by culturing E. col i in an LB medium including 50 pg/ ml of kanamyci n and addi ng ar abi nose when the absorbance at 600 nm reached 0.7 to induce the expression of the recombi nant prot ei n. Then, the cel I s i nduced to express the recombi nant protein were I ysed and filtered, and the target protei n was captured by usi ng a cat i on exchange method, an affinity column, and the like to obtain a highly purified recombi nant stabilized gal ect i n 9 protein in a high yield.
<Exampl e 1> Conf i r mat i on of Ti m-3 Protein Binding Ability of Recombi nant St abl e Gal ect i n 9 Pr ot ei n ( sGal -9) 100 pl of recombinant quman im-3 (R&D systems, Cat.
No. 10241-1I-050) at a concentration of 0.5 pg/ml was added to each well of a 96-well ELI SA plate (Nunc, Cat.
No. 44-2404-21), the reaction was carried out at room temperature for one night, and then the resultant was washed three times with DPBS (Dulbecco's phosphate-buffered saline; Wel gene, Cat. No. LB011-02) including 0.05% Tween 20 (Sigma Cat. No. P9416). After washing, 200 pl of DPBS including 1% bovine serum albumin (BSA) was added to each well, incubated at room temperature for one hour, and washed three times with a wash buffer. After washing three times, the recombinant stabilized galectin 9 protein (sGal-9) obtained in <Preparation Example 1>
was diluted to various concentrations (0, 5, 10 and 20 pg/ml) and added by 100 pl to each well, incubated at room temperature for two hours, and washed three times with a wash buffer.
For an experimental group, Gal-9 antibody (R&D
systems, Cat. No. 1015238) was diluted to 1:1000 by using 1% BSA/DP3S and added by 100 n1 to eacq well, incubated at room temperature for one hour, and washed three times with a wash buffer. As a control group, an anti-mouse HRP
antibody (lnvitrogen, Cat. No. 31430) was diluted to 1:1000 by using 1% BSA/DPBS and added by 100 pl to each well, incubated at room temperature for one hour, and washed three times with a wash buffer.
100 pl of a 1:1 mixture of substrate reagents (R&D
systems, Cat. No. DY999) A and B was added to each washed well and incubated at room temperature with the light blocked for 20 minutes. After the incubation, 50 pl of a stop solution (2 N H2SO4) was added to each well, and the optical density (0.D) value was measured and checked at 450 nm using a microplate reader.
As a result, as shown in FIG. 1, it was confirmed that the optical density increased as the concentration of sGal -9 i ncr eased, and thus it was conf i rmed that the binding degree of sGal - 9 and Ti m- 3 was excellent in a concent r at i on-dependent manner ( Fl G. 1) .
<Exampl e 2> Conf i r mat i on of T Hel per Cell Differentiation Inhibitory Effect of sGal - 9 Anti - CD3 ( eBi osci ence, Cat. No. 16-0031-82) at a concentration of 2 pg/m1 diluted with PBS (Welgene, Cat.
No. LB 001-02) was added to a 96-well plate by 100 p1 or to a 24-well plate by 500 u11 was reacted at 4 C for one ni ght, was washed once with PBS, and was used for cel 1 culture. After the resultant was dispensed into 96-well pl at es or 24- wel 1 pl at es coated with CD4, CD62L and T
cells and incubated at 37 C and 5% CO2 condi ti ons for four days, ant i -1 L-4, ant i - I L-2 and ant i -1 L-12 cyt oki nes were added to i nduce the di f f erent i at i on i nt o Th1 cell s and anti-IFN-y, anti-IL-4, anti-IL-2 and anti-IL-6 cytokines were added to i nduce di f f erent i at i on i nto Th17 cells.
After the addition of cytoki nes, sGal - 9 with a concentration of 1 lag/m1 was added and incubated at 37 C
and 5% CO2 condi ti ons for four days.
After the culture, IFN-y cells of ml cells and IL-17 cytoki nes of Th17 cells were measured by usi ng an ELI SA assay kit ( Mouse DuoSet, R&D Systems) according to the procedure of the manufacturer.
As a result, as shown in FIG. 2, it was confirmed that the di f f erent i at i on of Th1 and Th17 cell s whi ch are the helper T cells was inhibited as the concentration of sGal - 9 increased, and thus it was confirmed that the hel per T cell differentiation inhibitory effect of sGal -9 was excellent ( Fl G. 2) .
<Exampl e 3> Conf i r mat i on of Fi brobl ast- I i ke-synovi ocyt es ( FLS) Apoptosi s Effect of sGal - 9 200 pl of the FLS cells (cell passage: RA
_______________________________________________________________ FLS 2-92#P7) were di spensed i nto each well of a 48- wel I pl ate (SPL, Cat. No. 30048) at a cell density of 5 x 104 cells/ml, and after 24 hours, 100 pl of sGal-9 at various concentrations (2.5, 5, and 10 pg/ml) was added to each wel I . After 16 hours, the supernatant was col I ect ed and stored in a deep freezer.
Cell s for fl uorescence act i vat ed cell sort i ng ( FACS) were placed and washed in a 15 ml tube, 200 pl of FACS
buffer was added thereto, and the resultant was stored at 4 C for one hour and was stai ned with Annexi n V- Fl TC/ 7-AAD ( Bi ol egend, Cat. No. 640922) . The stai ned cells were filtered witq a 70 pm cell strainer (3D Falcon, Cat. No.
352350) into a tube for FACS.
As a result, as shown in FIG. 3, it was confirmed that the f i brobl ast - I i ke- synovi ocyt es apopt osi s effect i ncr eased as the concent r at i on of sGal -9 i ncr eased, and thus it was conf i rmed that the synovi al cell apoptosi s effect of sGal - 9 was excellent ( Fl G. 3) .
<Exampl e 4> Conf i r mat i on of Osteocl ast Di f f erent i at i on Inhibitory Effect of sGal - 9 1 ml of osteocl asts was passaged i n each wel I of a 12- wel I pl ate ( Corni ng, Cat. No. 3513) at a cell densi ty of 2.5 x 105 cel I s/ ml , and after 24 hours, sGal - 9 as an exper i mental group at van i ous concent r at i ons (0. 1, 0. 5, 1, 2, 4, and 10 ag/m1)and PBS as a control group were put into each well by 1 ml.
After compl et i on of di f f er ent i at i on, the stai ned osteocl asts of the experimental group and the control group were TRAP- stai ned by usi ng the TRAP St ai ni ng Kit ( K- ASSAY, Cat. No. KT-008) and counted by using an opt i cal mi croscope, and then multi nucl eat ed cel I s (TRAP( +) MNCs) i n whi ch three or more nucl ei were observed was determined to be differentiated into osteocl asts.
As a result, as shown in FIG. 4, it was confirmed that the number of stai ned osteocl asts decreased as the concent r at i on of sGal -9 i ncr eased, and thus it was conf i rmed that the osteocl ast differentiation inhibitory effect of sGal -9 was excellent ( Fl G. 4).
<Exampl e 5> Conf i r mat i on of Bone Resorpt i on I nhi bi tory Effect of sGal -9 1 ml of osteocl asts was passaged at a cel I densi ty of 2.5 x 105 cell s/ ml into each well of Osteo Assay Surf ace multi-well plate ( Corni ng, Cat. No. 3987) coated with i nor gani c three-di mensi onal crystal I i ne that resembled the bone structure in vi vo, and after 24 hours, sGal -9 as an experi mental group at van i ous concent r at i ons (0.1, 0.5, 1, 2, /, and 10 pg/m1)and PBS as a control group were put i nto each wel I by 1 ml . The experi mental group ( sGal -9), the control ( PBS), and cell culture medium ( MEM-al pha, gi bco, Cat. No. 12561-056) were repl aced daily unt i I di f f erent i at i on was compl et ed.
After completion of differentiation, cells were removed with 20% SOS (Bleaching solution), and the Osteo Assay Surf ace multi -wel I pl ate was observed under a mi croscope.
As a result, as shown in FIG. 5, it was confirmed that the bone resorpti on inhibitory effect of sGal -9 was excellent ( Fl G. 5).
I n concl usi on, through the results of <Exampl e 1> to <Exampl e 5>, it was confirmed that bone diseases could be prevented or treated by sGal -9 i n a concent rat i on-dependent manner, through the i ncrease i n the Ti m-3 protei n bi ndi ng ability, the i ncreases i n the Thl and Th17 cell di f f erent i at i on i nhi bi t or y effect and the synovi al cel I apoptosi s effect, and the i ncr eases i n the ost eocl ast di f f erent i at i on i nhi bi t or y effect and the bone r esor pt i on i nhi bi t or y effect.
Claims
[Cl ai ms]
[Cl ai m 1]
A recombi nant stabi l i zed gal ecti n 9 protei n havi ng:
an ami no aci d sequence represented by SEQ l D NO: 1.
[C1 ai m 2]
The recombi nant stabi l i zed gal ecti n 9 protei n accordi ng to cl ai m 1, wherei n the recombi nant stabi l i zed gal ecti n 9 protei n mai ntai ns a sugar chai n recogni ti on act i vi ty of wi l d-type gal ecti n 9.
[C1 ai m 3]
A recombi nant vector compri si ng:
a gene that encodes the protei n of cl ai m 1.
[C1 ai m 4]
A transf ormant i nto whi ch the recombi nant vector of cl ai m3isi nserted.
[C1 ai m 5]
A pharmaceuti cal composi ti on f or prevent i on or treatment of a bone di sease, compri si ng:
a recombi nant stabi l i zed gal ecti n 9 protei n havi ng an ami no aci d sequence represented by SEQ l D NO: 1 or a pol ynucl eoti de that encodes the same, as an act i ve i ngredi ent.
[C1 ai m 6]
The pharmaceuti cal composi ti on f or prevent i on or treatment of a bone di sease accordi ng to cl ai m 5, wherei n the recombi nant stabi l i zed gal ecti n 9 prot ei n exhi bi ts one or more of t he f ol l owi ng propert i es:
i ) i ncr ease i n a degree of bi ndi ng Ti m- 3 protei n;
i i ) i nhi bi ti on of Th1 and Th17 cel l di f f erenti at i on;
iii) i nhi bi ti on of T hel per cel I s;
i v) apoptosi s of f i brobl ast- l i ke synovi ocyt es ( FLS);
and v) i nhi bi ti on of osteocl ast di f f erenti at i on and bone r esor pt i on.
[C1 ai m 7]
The pharmaceuti cal composi ti on f or prevent i on or treatment of a bone di sease accordi ng to cl ai m 5, wherei n the bone di sease i s at l east one sel ected f rom the group consi sti ng of art hri ti s, osteoporosi s, per i prost het i c osteol ysi s, osteol ysi s caused by f at i gue debri s of i mpl ants, Paget' s di sease, osteomal aci a, ri cket s, osteopeni a, cal ci um dysregul at i on, a met ast at i c bone cancer, an i nf I ammatory bone l oss, a secondary bone l oss due to an endocri ne di sease or drugs, and per i odont al di seases accompani ed by dest r uct i on of al veol ar bone.
[C1 ai m 8]
The pharmaceuti cal composi ti on f or prevent i on or treatment of a bone di sease accordi ng to cl ai m 7, wherei n the art hritis is sel ected f rom the group consi st i ng of ost eoart hr i t i s, degener at i ve art hr i t i s, rheumatoi d art hri ti s, di ssoci at i ve osteochondri ti s, j oi nt l i gament damages, meni scus damages, j oi nt mi sal i gnment, avascul ar necrosi s, and j uveni l e i di opat hi c art hri ti s.
[C1 ai m 9]
The pharmaceuti cal composi ti on f or prevent i on or treatment of bone di sease accordi ng to cl ai m 5, wherei n the bone di sease i s rheumatoi d arthritis or ost eopor osi s.
[C1 ai m 10]
The pharmaceuti cal composi ti on f or prevent i on or treatment of a bone di sease accordi ng to cl ai m 5, wher ei n t he pharmaceut i cal composi t i on f urt her compri ses a pharmaceuti cal l y acceptabl e carri er.
[C1 ai m 11]
The pharmaceuti cal composi ti on f or prevent i on or treatment of a bone di sease accordi ng to cl ai m 5, wherei n t he pharmaceut i cal composi t i on i s f or mul at ed f or oral admi ni strati on, i ntramuscul ar admi ni strati on, i nt r avenous admi ni st r at i on, i nt raper i t oneal admi ni st r at i on, subcut aneous admi ni st r at i on, i nt r ader mal admi ni strati on, or topi cal admi ni strati on.
[C1 ai m 12]
A heal th f uncti onal f ood f or pr event i on or i mprovement of a bone di sease, compri si ng:
a recombi nant stabi I i zed gal ecti n 9 protei n havi ng an ami no aci d sequence represented by SEQ I D NO: 1, as an act i ve i ngredi ent.
[C1 ai m 13]
A method f or treati ng bone di sease, compri si ng:
admi ni steri ng to an i ndi vi dual a pharmaceuti cal I y eff ecti ve amount of a recombi nant stabi I i zed gal ecti n 9 protei n havi ng an ami no aci d sequence represented by SEQ
I D NO: 1 or a pol ynucl eoti de that encodes the same.
[C1 ai m 14]
A method f or prevent i ng or i mprovi ng a bone di sease, compri si ng:
admi ni steri ng to an i ndi vi dual a pharmaceuti cal I y eff ecti ve amount of a recombi nant stabi I i zed gal ecti n 9 protei n havi ng an ami no aci d sequence represented by SEQ
I D NO: 1 or a pol ynucl eoti de that encodes the same.
[C1 ai m 15]
A pharmaceuti cal composi ti on used f or pr event i on or treatment of a bone di sease, compri si ng:
a recombi nant stabi I i zed gal ecti n 9 protei n havi ng an ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eoti de that encodes the same.
[C1 ai m 16]
A heal th f uncti onal f ood used f or prevent i on or i mprovement of a bone di sease, compri si ng:
a recombi nant stabi l i zed gal ecti n 9 protei n havi ng an ami no aci d sequence represented by SEQ l D NO: 1 or a pol ynucl eoti de that encodes the same.
[C1 ai m 17]
A use of a recombi nant stabi l i zed gal ecti n 9 protei n havi ng an ami no aci d sequence represented by SEQ l D NO: 1 or a pol ynucl eoti de that encodes the same f or prepar at i on of a pharmaceuti cal composi ti on f or pr event i on or treatment of a bone di sease.
[C1 ai m 18]
A use of a recombi nant stabi l i zed gal ecti n 9 protei n havi ng an ami no aci d sequence represented by SEQ l D NO: 1 or a pol ynucl eoti de that encodes the same f or prepar at i on of a heal t h f unct i onal f ood f or prevent i on or i mprovement of a bone di sease.
[Cl ai m 1]
A recombi nant stabi l i zed gal ecti n 9 protei n havi ng:
an ami no aci d sequence represented by SEQ l D NO: 1.
[C1 ai m 2]
The recombi nant stabi l i zed gal ecti n 9 protei n accordi ng to cl ai m 1, wherei n the recombi nant stabi l i zed gal ecti n 9 protei n mai ntai ns a sugar chai n recogni ti on act i vi ty of wi l d-type gal ecti n 9.
[C1 ai m 3]
A recombi nant vector compri si ng:
a gene that encodes the protei n of cl ai m 1.
[C1 ai m 4]
A transf ormant i nto whi ch the recombi nant vector of cl ai m3isi nserted.
[C1 ai m 5]
A pharmaceuti cal composi ti on f or prevent i on or treatment of a bone di sease, compri si ng:
a recombi nant stabi l i zed gal ecti n 9 protei n havi ng an ami no aci d sequence represented by SEQ l D NO: 1 or a pol ynucl eoti de that encodes the same, as an act i ve i ngredi ent.
[C1 ai m 6]
The pharmaceuti cal composi ti on f or prevent i on or treatment of a bone di sease accordi ng to cl ai m 5, wherei n the recombi nant stabi l i zed gal ecti n 9 prot ei n exhi bi ts one or more of t he f ol l owi ng propert i es:
i ) i ncr ease i n a degree of bi ndi ng Ti m- 3 protei n;
i i ) i nhi bi ti on of Th1 and Th17 cel l di f f erenti at i on;
iii) i nhi bi ti on of T hel per cel I s;
i v) apoptosi s of f i brobl ast- l i ke synovi ocyt es ( FLS);
and v) i nhi bi ti on of osteocl ast di f f erenti at i on and bone r esor pt i on.
[C1 ai m 7]
The pharmaceuti cal composi ti on f or prevent i on or treatment of a bone di sease accordi ng to cl ai m 5, wherei n the bone di sease i s at l east one sel ected f rom the group consi sti ng of art hri ti s, osteoporosi s, per i prost het i c osteol ysi s, osteol ysi s caused by f at i gue debri s of i mpl ants, Paget' s di sease, osteomal aci a, ri cket s, osteopeni a, cal ci um dysregul at i on, a met ast at i c bone cancer, an i nf I ammatory bone l oss, a secondary bone l oss due to an endocri ne di sease or drugs, and per i odont al di seases accompani ed by dest r uct i on of al veol ar bone.
[C1 ai m 8]
The pharmaceuti cal composi ti on f or prevent i on or treatment of a bone di sease accordi ng to cl ai m 7, wherei n the art hritis is sel ected f rom the group consi st i ng of ost eoart hr i t i s, degener at i ve art hr i t i s, rheumatoi d art hri ti s, di ssoci at i ve osteochondri ti s, j oi nt l i gament damages, meni scus damages, j oi nt mi sal i gnment, avascul ar necrosi s, and j uveni l e i di opat hi c art hri ti s.
[C1 ai m 9]
The pharmaceuti cal composi ti on f or prevent i on or treatment of bone di sease accordi ng to cl ai m 5, wherei n the bone di sease i s rheumatoi d arthritis or ost eopor osi s.
[C1 ai m 10]
The pharmaceuti cal composi ti on f or prevent i on or treatment of a bone di sease accordi ng to cl ai m 5, wher ei n t he pharmaceut i cal composi t i on f urt her compri ses a pharmaceuti cal l y acceptabl e carri er.
[C1 ai m 11]
The pharmaceuti cal composi ti on f or prevent i on or treatment of a bone di sease accordi ng to cl ai m 5, wherei n t he pharmaceut i cal composi t i on i s f or mul at ed f or oral admi ni strati on, i ntramuscul ar admi ni strati on, i nt r avenous admi ni st r at i on, i nt raper i t oneal admi ni st r at i on, subcut aneous admi ni st r at i on, i nt r ader mal admi ni strati on, or topi cal admi ni strati on.
[C1 ai m 12]
A heal th f uncti onal f ood f or pr event i on or i mprovement of a bone di sease, compri si ng:
a recombi nant stabi I i zed gal ecti n 9 protei n havi ng an ami no aci d sequence represented by SEQ I D NO: 1, as an act i ve i ngredi ent.
[C1 ai m 13]
A method f or treati ng bone di sease, compri si ng:
admi ni steri ng to an i ndi vi dual a pharmaceuti cal I y eff ecti ve amount of a recombi nant stabi I i zed gal ecti n 9 protei n havi ng an ami no aci d sequence represented by SEQ
I D NO: 1 or a pol ynucl eoti de that encodes the same.
[C1 ai m 14]
A method f or prevent i ng or i mprovi ng a bone di sease, compri si ng:
admi ni steri ng to an i ndi vi dual a pharmaceuti cal I y eff ecti ve amount of a recombi nant stabi I i zed gal ecti n 9 protei n havi ng an ami no aci d sequence represented by SEQ
I D NO: 1 or a pol ynucl eoti de that encodes the same.
[C1 ai m 15]
A pharmaceuti cal composi ti on used f or pr event i on or treatment of a bone di sease, compri si ng:
a recombi nant stabi I i zed gal ecti n 9 protei n havi ng an ami no aci d sequence represented by SEQ I D NO: 1 or a pol ynucl eoti de that encodes the same.
[C1 ai m 16]
A heal th f uncti onal f ood used f or prevent i on or i mprovement of a bone di sease, compri si ng:
a recombi nant stabi l i zed gal ecti n 9 protei n havi ng an ami no aci d sequence represented by SEQ l D NO: 1 or a pol ynucl eoti de that encodes the same.
[C1 ai m 17]
A use of a recombi nant stabi l i zed gal ecti n 9 protei n havi ng an ami no aci d sequence represented by SEQ l D NO: 1 or a pol ynucl eoti de that encodes the same f or prepar at i on of a pharmaceuti cal composi ti on f or pr event i on or treatment of a bone di sease.
[C1 ai m 18]
A use of a recombi nant stabi l i zed gal ecti n 9 protei n havi ng an ami no aci d sequence represented by SEQ l D NO: 1 or a pol ynucl eoti de that encodes the same f or prepar at i on of a heal t h f unct i onal f ood f or prevent i on or i mprovement of a bone di sease.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR20190143752 | 2019-11-11 | ||
| KR10-2019-0143752 | 2019-11-11 | ||
| PCT/KR2020/015782 WO2021096217A1 (en) | 2019-11-11 | 2020-11-11 | Pharmaceutical composition, comprising recombinant stabilized galectin 9 protein, for prevention or treatment of rheumatoid arthritis and bone disease |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA3157954A1 true CA3157954A1 (en) | 2021-05-20 |
Family
ID=76142932
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA3157954A Pending CA3157954A1 (en) | 2019-11-11 | 2020-11-11 | Pharmaceutical composition, comprising recombinant stabilized galectin 9 protein, for prevention or treatment of rheumatoid arthritis and bone disease |
Country Status (4)
| Country | Link |
|---|---|
| KR (1) | KR102267413B1 (en) |
| CN (1) | CN115605217A (en) |
| BR (1) | BR112022005782A2 (en) |
| CA (1) | CA3157954A1 (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8268324B2 (en) * | 2004-03-29 | 2012-09-18 | Galpharma Co., Ltd. | Modified galectin 9 proteins and use thereof |
| US9908921B2 (en) * | 2012-11-20 | 2018-03-06 | National University Corporation Kagawa University | Modified galectin-9 protein |
-
2020
- 2020-11-10 KR KR1020200149295A patent/KR102267413B1/en active IP Right Grant
- 2020-11-11 BR BR112022005782A patent/BR112022005782A2/en unknown
- 2020-11-11 CN CN202080077890.XA patent/CN115605217A/en active Pending
- 2020-11-11 CA CA3157954A patent/CA3157954A1/en active Pending
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| BR112022005782A2 (en) | 2022-10-11 |
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| KR20210056927A (en) | 2021-05-20 |
| KR102267413B1 (en) | 2021-06-21 |
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