WO2023033130A1 - Composition for treating or preventing bone diseases - Google Patents

Abstract

The present inventors found that secretory Siglec-9 inhibits the differentiation of bone marrow cells and macrophages into osteoclasts and increases bone density, and that secretory Siglec-9 inhibits the inflammatory reaction of fibroblasts. Therefore, the present disclosure provides a composition that is for treating or preventing bone diseases, and that contains secretory Siglec-9. In another embodiment, the present disclosure provides a composition that is for increasing bone density, and that contains secretory Siglec-9. In further another embodiment, the present disclosure provides a composition that is for preventing fracture, and that contains secretory Siglec-9. In further another embodiment, the present disclosure provides a composition that is for inhibiting differentiation into osteoclasts, and that contains secretory Siglec-9. In further another embodiment, the present disclosure provides a composition that is for inhibiting the inflammatory reaction of fibroblasts, and that contains secretory Siglec-9.

Description

COMPOSITIONS FOR TREATMENT OR PREVENTION OF BONE DISEASES

This patent application claims priority from Japanese Patent Application No. 2021-144208, which is incorporated herein in its entirety by reference.
The present application relates to compositions for treating or preventing bone disease.

　Rheumatoid arthritis is an autoimmune disease of unknown cause characterized by protracted destructive arthritis. Treatment with methotrexate and targeted drugs (biologics and JAK inhibitors) is standard practice. On the one hand, rheumatoid arthritis is a risk factor for osteoporosis. The prevalence of osteoporosis in rheumatoid arthritis patients is about twice that of controls, and fracture risk is also significantly higher. Conventional therapeutic drugs for rheumatoid arthritis, including biologics and JAK inhibitors, do not have the effect of improving bone density, and therapeutic agents for osteoporosis, for example, organisms that inhibit RANKL, an osteoclast-inducing factor, are used as necessary. drug (denosumab) has been used.

The use of secretory sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) is expected as a therapeutic agent for rheumatoid arthritis. Siglec-9 is a type I transmembrane receptor protein expressed in monocytes, granulocytes and macrophages, with extracellular, transmembrane and cytoplasmic domains. Secretory Siglec-9 is a form of Siglec-9 lacking the cytoplasmic domain. When secretory Siglec-9 alone is intravenously administered to collagen-induced arthritis model mice, secretory Siglec-9 is induced in injured joints. It accumulated and improved the pathology of rheumatoid arthritis (Patent Document 1, Non-Patent Document 1).

International Publication No. 2014/098249 pamphlet

An object of the present disclosure is to provide new means for treating or preventing bone diseases.

In studying the therapeutic effects of secretory Siglec-9 in rheumatoid arthritis, the present inventors found that secretory Siglec-9 inhibits the differentiation of bone marrow cells and macrophages into osteoclasts and increases bone density. , and suppressed the inflammatory response of fibroblasts.

Accordingly, in certain aspects, the present disclosure provides compositions for treating or preventing bone disease comprising secreted Siglec-9.

In yet another aspect, the present disclosure provides compositions for increasing bone density comprising secreted Siglec-9.

In yet another aspect, the present disclosure provides compositions for preventing bone fractures comprising secreted Siglec-9.

In yet another aspect, the present disclosure provides a composition for suppressing osteoclast differentiation, comprising secreted Siglec-9.

In yet another aspect, the present disclosure provides compositions for suppressing fibroblast inflammatory responses comprising secreted Siglec-9.

According to the present disclosure, compositions for treating or preventing bone disease, compositions for increasing bone density, compositions for preventing fractures, compositions for inhibiting osteoclast differentiation, or fibroblast Compositions are provided for inhibiting cellular inflammatory responses.

1 shows the experimental schedule of Test 1 using anti-collagen antibody arthritis (CAIA) model mice and the therapeutic effects of sSiglec-9 and JAKi on arthritis. 7 shows the bone area and bone density of the cuboid bone 7 days after the start of treatment intervention in Test 1. FIG. 7 shows the results of hematoxylin and eosin staining of ankle joints on day 7 from the start of therapeutic intervention in Test 1. FIG. 4 is an enlarged view within the frame of FIG. 3; FIG. 7 shows the scores of ankle bone erosion and cartilage destruction on day 7 from the start of treatment intervention in Study 1. FIG. 7 shows the number of cells expressing markers related to inflammation or bone destruction in ankle joints on day 7 from the start of therapeutic intervention in Study 1. FIG. Gene expression of cartilage degrading enzymes and inflammatory markers in synovial fibroblasts stimulated with TNFα in the presence or absence of sSiglec-9. Mouse bone marrow cells were induced to differentiate into osteoclasts with MCSF and RANKL in the presence or absence of sSiglec-9, and the results of measuring the number of TRAP ⁺ cells and viable cells are shown. Mouse bone marrow cells (mBM) or RAW264.7 macrophages were induced to differentiate into osteoclasts with MCSF and RANKL in the presence or absence of sSiglec-9, and the gene expression of TRAP and cathepsin K (ctsk) was measured. indicate. The experimental schedule of Test 4 using osteoporosis model mice (OVX mice) is shown. FIG. 10 is an image of the lower extremities taken 28 days after ovariectomy in Test 4 using a μCT imaging device. FIG. Bone parameters of the femur 28 days after ovariectomy in study 4 (ratio of cancellous bone volume to intramedullary tissue volume, average trabecular width, average trabecular number per unit length, trabecular gap). .

Unless otherwise specified, the terms used in this specification have the meanings commonly understood by those skilled in the art in fields such as organic chemistry, medicine, pharmacy, molecular biology, and microbiology. Listed below are definitions for some terms used herein, which definitions supersede general understanding herein.

In this specification, when a numerical value is accompanied by the term "about", it is intended to include a range of ±10% of that value. For example, "about 20" shall include "18-22". Numerical ranges include all numbers between and including the endpoints. "About" in reference to a range applies to both endpoints of that range. Thus, for example, "about 20 to 30" shall include "18 to 33."

Sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) is a transmembrane protein expressed in monocytes, granulocytes and macrophages, and homologues are known in humans and various animals. In the present disclosure, Siglec-9 can be of any species, typically mammals (e.g., humans, dogs, cats, rabbits, cows, pigs, goats, sheep, horses, monkeys, guinea pigs, rats). and mice), preferably human.

Siglec-9 typically contains a signal peptide, an extracellular domain, a transmembrane domain and an intracellular domain, and soluble proteins containing the extracellular domain of Siglec-9 also exist in vivo. In the present disclosure, "secreted Siglec-9" refers to a protein that contains the extracellular domain of Siglec-9 and does not contain the transmembrane and intracellular domains. The secreted Siglec-9 may further comprise a Siglec-9 signal peptide.

The amino acid sequences of Siglec-9 derived from various species can be easily obtained using publicly known databases. A representative amino acid sequence of human Siglec-9 is shown in SEQ ID NO:1. It consists of 463 amino acids, positions 1-17 are the signal peptide, positions 18-348 are the extracellular domain, positions 349-369 are the transmembrane domain, positions 370-463. The position is the intracellular domain. Thus, typically a secreted Siglec-9 comprises the amino acid sequence from positions 18 to 348 of SEQ ID NO:1 (SEQ ID NO:2). Secreted Siglec-9 may further comprise a signal peptide and the amino acid sequence of positions 1 to 348 of SEQ ID NO: 1 (SEQ ID NO: 3).

Secretory Siglec-9 includes a sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 or 3, as long as the activity of secretory Siglec-9 is maintained. You can The term "several" means preferably 2 to 7, more preferably 2 to 5, and most preferably 2 to 3 amino acids. Amino acid substitutions are preferably conservative substitutions between similar amino acid residues.

As long as the activity of secretory Siglec-9 is maintained, secretory Siglec-9 has the amino acid sequence of SEQ ID NO: 2 or 3 when calculated using BLAST or the like (for example, the parameters of the initial conditions of BLAST are at least about 70%, preferably at least about 80%, more preferably at least about 90%, particularly preferably at least about 95%, most preferably at least about 97%, about 98% or about 99% It may contain amino acid sequences with identity.

In the present disclosure, amino acid sequence identity means the degree of sequence similarity between proteins, and two proteins aligned in an optimal state (maximum amino acid match) over the region of the sequences to be compared. Determined by comparing sequences. A sequence identity number (%) is determined by determining the number of identical amino acids present in both sequences to determine the number of matching sites, then dividing this number of matching sites by the total number of amino acids in the sequence region being compared. , is calculated by multiplying the resulting value by 100. Algorithms for obtaining optimal alignment and sequence identity include various algorithms commonly available to those of skill in the art (eg, BLAST algorithms, FASTA algorithms, etc.). Sequence identity can be determined, for example, using sequence analysis software such as BLAST, FASTA.

The secretory Siglec-9 may be a fusion protein with any protein or polypeptide as long as the activity of the secretory Siglec-9 is maintained. Here, the proteins or polypeptides constituting the fusion protein include, for example, Fc region, histidine tag, GST tag, GFP tag, myc tag, FLAG tag and the like. A protein or polypeptide that constitutes a fusion protein may be attached to the extracellular domain of Siglec-9 via a linker. In some embodiments, the secreted Siglec-9 comprises an Fc region.

In the present disclosure, "secretory Siglec-9 activity" includes at least the activity of suppressing the differentiation of bone marrow cells or macrophages into osteoclasts. This activity can be measured by various methods known in the art. For example, it is known that bone marrow cells or macrophages can be stimulated with macrophage colony-stimulating factor (M-CSF) and NF-κB-activating receptor ligand (RANKL) to induce osteoclast differentiation. By inducing the differentiation of bone marrow cells or macrophages into osteoclasts and measuring the number of obtained osteoclasts, the activity of the protein to suppress differentiation into osteoclasts can be evaluated. The number of osteoclasts can be determined based on the expression of osteoclast markers such as TRAP or Cathepsin K. For example, it can be determined by adding a chromogenic substrate for the TRAP enzyme to cultured cells and evaluating the enzymatic activity (TRAP staining). Alternatively, the expression level of each marker can be measured by an immunological method, a quantitative real-time PCR method, or the like.

"Activity of secretory Siglec-9" may further include activity to suppress inflammation. This activity can be measured by various methods known in the art. For example, it is known that stimulation of fibroblasts such as synovial fibroblasts with TNFα, an inflammatory cytokine, enhances the expression of inflammatory markers. The anti-inflammatory activity of the protein can be assessed based on the stimulation and expression of inflammatory markers. Known inflammatory markers include, for example, iNos, IL-1β, IL-6, and TNFα. The expression level of each marker can be measured by an immunological method, a quantitative real-time PCR method, or the like.

Examples of immunological methods include flow cytometry analysis, radioisotope immunoassay (RIA method), enzyme immunoassay (ELISA method), western blotting, and immunohistochemical staining.

In the present disclosure, "the activity of secretory Siglec-9 is maintained" means that the activity of secretory Siglec-9 is maintained by about 30% or more, and may exceed 100%. It is preferably maintained at about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more.

Secretory Siglec-9 can be produced by known methods, such as genetic engineering techniques, for example, a method of producing an expression vector containing a polynucleotide encoding the same and expressing it in cells. Specifically, an expression vector is constructed such that a polynucleotide encoding a secretory Siglec-9 is expressed under an expression control region such as an enhancer or promoter, host cells are transformed with this expression vector, and secreted. Type Siglec-9 is expressed and recovered. Alternatively, commercially available secretory Siglec-9 may be used.

In the examples described later, secretory Siglec-9 improved bone area and bone density and suppressed bone destruction in rheumatoid arthritis models. It also inhibited osteoclast differentiation of myeloid cells and macrophages in vitro. Furthermore, it improved bone parameters and inhibited disease progression in an osteoporosis model. Therefore, secreted Siglec-9 may be used to treat or prevent bone disease. In the present disclosure, bone disease means a disease characterized by bone abnormalities. Bone abnormalities include, for example, increased or activated osteoclasts or decreased bone density. Bone diseases include osteoporosis, osteoarthritis and femoral head necrosis, especially osteoporosis.

As used herein, "treat a disease" or "treatment of a disease" means to alleviate, alleviate, ameliorate or eliminate a disease. As used herein, "prevent disease" or "prophylaxis of disease" is to prevent disease in a subject, particularly in a subject who may, but has not yet, developed the disease, or , means to reduce the likelihood of developing disease.

Subjects who may develop bone disease but have not yet developed it include, for example, subjects with decreased bone density or subjects with risk factors for bone disease. Risk factors for osteoporosis include, for example, aging, menopause, diabetes, chronic kidney disease, rheumatoid arthritis, hyperparathyroidism, hyperthyroidism, steroid drugs, and sex hormone lowering therapy. Risk factors for osteoarthritis include, for example, aging, genetics, occupation, obesity, cartilage fragility, trauma, joint dysplasia, joint lability. Risk factors for femoral head necrosis include, for example, steroid therapy and heavy alcohol consumption.

Rheumatoid arthritis is known as a risk factor for osteoporosis. In general, the initial symptoms of rheumatoid arthritis are mainly inflammation of the synovial membrane, and as it progresses, joint destruction occurs due to cartilage destruction and bone erosion. Further progression of rheumatoid arthritis can be complicated by osteoporosis near the joints (paraarticular osteoporosis) and systemic osteoporosis, especially in the elderly, and can increase the risk of fractures. Therefore, in the treatment of advanced rheumatoid arthritis, a therapeutic agent for rheumatoid arthritis and a therapeutic agent for osteoporosis have conventionally been used in combination. Secretory Siglec-9 can treat rheumatoid arthritis as described in Patent Document 1, and can treat and prevent osteoporosis as shown in the Examples of the present application. In such cases, there is no need to concomitantly use other osteoporosis therapeutic agents. Accordingly, in one embodiment, compositions are provided for treating or preventing osteoporosis in patients with rheumatoid arthritis, particularly advanced rheumatoid arthritis, comprising secreted Siglec-9. In another aspect, compositions for treating advanced rheumatoid arthritis, particularly rheumatoid arthritis with osteoporosis, comprising secretory Siglec-9 are provided.

As a classification of the progression of rheumatoid arthritis, for example, Stein blocker's staging is known. stage III (highly advanced stage) is a state in which bone and cartilage are destroyed; stage IV (end stage) is a state in which the joint is destroyed and movement is There is no state. In certain embodiments, the advanced rheumatoid arthritis is stage II-IV, stage III-IV, or stage IV rheumatoid arthritis according to the stain blocker staging system.

In one embodiment, the above-described treatment or prevention of bone disease or rheumatoid arthritis is characterized by not using agents that inhibit the activity of other osteoclasts. Other agents that inhibit osteoclast activity include, for example, bisphosphonate drugs (e.g. etidronate, alendronate, risedronate, minodronate, ibandronate), RANKL inhibitors (e.g. denosumab), sclerotin inhibitors (e.g. romosozumab). In one embodiment, the above treatment or prevention of bone disease or rheumatoid arthritis is characterized by the absence of secretory Siglec-9 in combination with monocyte chemoattractant-1 (MCP-1). In one embodiment, the above treatment or prevention of bone disease or rheumatoid arthritis is characterized by the use of secretory Siglec-9 as the sole active ingredient.

In another aspect, there is provided a composition for suppressing osteoclast differentiation, comprising secreted Siglec-9. The composition can be used in vivo to inhibit osteoclast differentiation in vivo. It can also be used in vitro by adding it to the culture medium.

In addition, since secretory Siglec-9 suppresses osteoclast differentiation, it can increase bone density and prevent fractures. Accordingly, in one aspect, compositions are provided for increasing bone density comprising secreted Siglec-9. Increasing bone density includes suppressing a decrease in bone density. In another aspect, compositions are provided for preventing bone fractures comprising secreted Siglec-9. These compositions can be used in accordance with compositions for treating or preventing bone disease.

In addition, in the examples described later, secretory Siglec-9 suppressed the inflammatory response of synovial fibroblasts in vitro. Accordingly, in one aspect, compositions are provided for suppressing fibroblast inflammatory responses comprising secreted Siglec-9. Examples of fibroblasts include synovial fibroblasts and periosteal fibroblasts. The composition can be used in vivo to suppress the inflammatory response of fibroblasts in vivo. It can also be used in vitro by adding it to the culture medium.

Targets to which secretory Siglec-9 is applied include mammals including humans (pets, livestock, experimental animals, etc.). Examples include humans, dogs, cats, rabbits, cows, pigs, goats, sheep, horses, monkeys, guinea pigs, rats and mice, especially humans. In one embodiment, the subject is a patient with rheumatoid arthritis, particularly advanced rheumatoid arthritis. In some embodiments, the subject is a human aged 60 or older, 65 or older, 70 or older, 75 or older, 80 or older, 85 or older, or 90 or older.

The composition of the present disclosure can contain bioabsorbable materials such as hyaluronic acid, collagen, fibrinogen, platelet plasma, and the like. Compositions of the present disclosure may also include gelling materials such as hyaluronic acid, collagen, fibrin glue, and the like. The compositions of the present disclosure can contain known pharmaceutically acceptable ingredients. For example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline and the like can be included. As these various additives, various known components can be appropriately used.

The formulation form of the composition of the present disclosure is not particularly limited. Various known formulation forms can be adopted. Tablets, powders, granules, granules, fine granules, capsules, solid injections that dissolve at the time of use, solids such as suppositories, liquid injections (intravenous/muscular injection), injections, drip infusion Examples include liquid agents such as formulations, eye drops, sprays, lotions, creams, topical agents such as patches, and the like. Moreover, it is possible to adopt a form in which it is carried by an indwelling type medical device or the like. Additionally, the compositions of the present disclosure can contain known pharmaceutically acceptable salts.

The dosage form of secretory Siglec-9 is not particularly limited. Various known dosage forms can be adopted depending on the application site and target disease. For example, parenteral administration may be systemic administration or local administration. More specific examples include injection, application or spraying to the diseased site. Intravenous administration, intraarterial administration, intraarticular administration, intraportal vein administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, intranasal administration, intraoral administration and the like can also be mentioned.

The dosage and administration of secretory Siglec-9 is not particularly limited. It can be set in consideration of the age, body weight, disease condition, etc. of the subject. For example, about 0.1 to about 1000 μg/kg body weight/day, about 1 to about 500 μg/kg body weight/day, about 10 to about 250 μg/kg body weight/day, or about 20 to about 100 μg/kg body weight/day. Secreted Siglec-9 may be administered.

The secretory Siglec-9 may be administered once, multiple times, or continuously. In the case of multiple administrations, for example, once to several times a day, for example, once, twice or three times a day, with an administration frequency of every day or every few days, for example, 1 day, 2 days, 3 days. Or it can be administered every 7 days. The administration period is not limited, and a drug holiday period may be provided.

In one aspect, a method of treating or preventing a bone disease is provided comprising administering secretory Siglec-9 to a subject in need of treatment or prevention of the bone disease.
In one aspect, secreted Siglec-9 for the treatment or prevention of bone disease is provided.
In one aspect, the use of secreted Siglec-9 for the treatment or prevention of bone disease is provided.
In one aspect, the use of secreted Siglec-9 in the manufacture of a composition for treating or preventing bone disease is provided.

In certain aspects, methods are provided for increasing bone density comprising administering secretory Siglec-9 to a subject in need thereof.
In one aspect, secreted Siglec-9 is provided for increasing bone density.
In one aspect, the use of secreted Siglec-9 to increase bone density is provided.
In one aspect, use of secreted Siglec-9 in the manufacture of a composition for increasing bone density is provided.

In certain aspects, methods are provided for preventing bone fractures comprising administering secreted Siglec-9 to a subject in need thereof.
In one aspect, secreted Siglec-9 is provided for preventing fractures.
In one aspect, the use of secreted Siglec-9 to prevent fractures is provided.
In one aspect, use of secreted Siglec-9 in the manufacture of a composition for preventing bone fracture is provided.

In one aspect, methods are provided for inhibiting osteoclast differentiation comprising administering a secreted form of Siglec-9 to a subject in need of inhibiting osteoclast differentiation.
In one aspect, methods are provided for inhibiting osteoclast differentiation comprising culturing cells in a culture medium containing secreted Siglec-9.
In one aspect, secreted Siglec-9 is provided for inhibiting osteoclast differentiation.
In one aspect, the use of secreted Siglec-9 to inhibit osteoclast differentiation is provided.
In one aspect, use of secreted Siglec-9 in the manufacture of a composition for inhibiting osteoclast differentiation is provided.

In one aspect, methods are provided for suppressing a fibroblast inflammatory response comprising administering a secreted Siglec-9 to a subject in need of suppressing the fibroblast inflammatory response.
In one aspect, a method is provided for suppressing a fibroblast inflammatory response comprising culturing cells in a culture medium containing secreted Siglec-9.
In one aspect, a secreted form of Siglec-9 is provided for suppressing a fibroblast inflammatory response.
In one aspect, the use of secreted Siglec-9 to suppress fibroblast inflammatory responses is provided.
In one aspect, use of secreted Siglec-9 in the manufacture of a composition for inhibiting fibroblast inflammatory response is provided.

The present disclosure provides, for example, the following embodiments.
[1] A composition for treating or preventing a bone disease, comprising secretory Siglec-9.
[2] The composition of item 1, wherein the bone disease is characterized by an increase or activation of osteoclasts or a decrease in bone density.
[3] The composition according to item 1, wherein the bone disease is osteoporosis, osteoarthritis or femoral head necrosis.
[4] The composition according to any one of items 1 to 3, wherein the bone disease is osteoporosis.
[5] The composition according to any one of items 1 to 4, for treating or preventing bone disease in patients with rheumatoid arthritis.
[6] The composition according to any one of items 1 to 5, for treating or preventing osteoporosis in rheumatoid arthritis patients.
[7] The composition according to any one of items 1 to 6, further for treating or preventing rheumatoid arthritis.
[8] The composition according to any one of items 5 to 7, wherein the rheumatoid arthritis is advanced rheumatoid arthritis.
[9] The composition according to any one of items 5 to 8, wherein the rheumatoid arthritis is stage II-IV, stage III-IV or stage IV rheumatoid arthritis according to stain blocker staging.
[10] The composition according to any one of items 5 to 9, further for preventing bone fractures in rheumatoid arthritis patients.
[11] A composition for increasing bone density, comprising secretory Siglec-9.
[12] A composition for preventing bone fracture, comprising secretory Siglec-9.
[13] A composition for suppressing osteoclast differentiation, comprising secretory Siglec-9.
[14] A composition for suppressing the inflammatory response of fibroblasts, comprising secretory Siglec-9.
[15] The composition according to any one of items 1 to 14, wherein the secreted Siglec-9 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:2 or 3.
[16] The composition according to any one of items 1 to 15, wherein the secretory Siglec-9 comprises the amino acid sequence of SEQ ID NO:2 or 3.
[17] The composition according to any one of items 1 to 16, wherein the secreted Siglec-9 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:2.
[18] The composition according to any one of items 1 to 17, wherein the secretory Siglec-9 comprises the amino acid sequence of SEQ ID NO:2.
[19] The composition according to any one of items 1 to 18, wherein the secretory Siglec-9 comprises an Fc region.

All documents cited herein are hereby incorporated by reference.
EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these Examples. Moreover, the above description is all non-limiting, and the present invention is defined in the appended claims, and various modifications are possible without departing from the technical idea thereof.

Test 1: sSiglec-9 suppresses bone destruction in rheumatoid arthritis model anti-collagen antibody arthritis (CAIA) model mice (T Kagari, H Doi and T Shimozato. The importance of IL-1β and TNF-α, and the noninvolvement of IL-6, in the development of monoclonal antibody-induced arthritis. J Immunol 169:1459-1466, 2002) was used. Joint destruction progresses when the inflammatory state of CAIA is at its highest level and continues for 7 days. From the late stage of joint destruction (15 days after arthritis-inducing antibody administration), 50 μg/kg body weight of sSiglec-9 (R & D SYSTEMS, Recombinant Human Siglec-9 Fc Chimera) was administered twice to the tail vein (15th and 17th days). Alternatively, JAKi (tofacitinib, 15 mg/kg body weight), an existing therapeutic agent for rheumatoid arthritis, was orally administered for 6 consecutive days. sSiglec-9 is a fusion protein consisting of the extracellular domain of human Siglec-9 (SEQ ID NO:2), a linker (SEQ ID NO:4) and the Fc region of human _IgG1 .

We continuously evaluated the extremity arthritis score. The results are shown in FIG. The effect of suppressing arthritis was similar between the sSiglec-9 group and the JAKi group.

　On the 7th day after the start of treatment intervention (20th day after the administration of arthritis-inducing antibody), the ankle joint was collected and the structural changes of the plantar bone were observed by microCT analysis. Since the cuboid is relatively large, measurement errors are unlikely to occur, and not only bone destruction due to intertarsal arthritis but also the metatarsal-tarsal joint can be evaluated at the same time. Density (relative CT value) was evaluated. The results are shown in FIG. Bone area and bone density were significantly improved in the sSiglec-9 group compared to the PBS group and JAKi group before (14th day) and after (20th day) the intervention.

On the 7th day after the start of treatment intervention, the ankle joint was collected and histologically analyzed. FIG. 3 shows the results of hematoxylin-eosin staining. FIG. 4 is an enlarged image within the frame of FIG. After the therapeutic intervention, severe proliferation of synovial cells, malformation, and bone resorption were observed around the cuboid bone in the PBS group. Synovial membrane activation and bone resorption were also observed in some JAKi groups. On the other hand, the sSiglec-9 group maintained the morphology of bone and cartilage and improved the synovial morphology. The synovial membrane of the sSiglec-9 group exhibited good granulation-like histology and was rich in blood vessels.

Histological staining images were observed, and bone erosion and cartilage destruction near the pannus were scored according to the criteria shown in the table below. The results are shown in FIG. Bone erosion and cartilage destruction were significantly improved in the sSiglec-9 group compared to the PBS and JAKi groups after intervention (20 days).

Ankle joints were collected on the 7th day from the start of therapeutic intervention, and the JAK activity marker (pSTAT3) around the cuboid, inflammatory markers (IL-1β, TNF-α), osteoclast marker (TRAP), osteoblast marker ( Osterix) and chondroblast marker (SOX9) expression changes were assessed histochemically by immunostaining. The results are shown in FIG. A decrease in the number of pSTAT3+ cells was confirmed in the JAKi group and the sSiglec-9 group, indicating that JAK activity was sufficiently suppressed in this experimental protocol. Notably, not only did IL-1β ⁺ cells, TNFα ⁺ cells and TRAP ⁺ osteoclast numbers decrease in the sSiglec-9 group, but also Osterix ⁺ osteoblast and SOX9 ⁺ chondroprogenitor cell numbers increased, It was inferred that the joint changed from a destructive environment to a regenerative environment.

Test 2: sSiglec-9 suppresses the inflammatory response of synovial fibroblasts Collecting synovial fibroblasts (SF) from the ankle joint of healthy mice It was stimulated with the cytokine TNFα. Gene expression of cartilage-degrading enzymes and inflammatory markers was assessed using quantitative real-time PCR methods. The results are shown in FIG. TNFα stimulation increased the gene expression of the cartilage degrading enzyme MMP9 and the inflammatory markers iNos, IL-1β, IL-6, and TNFα, but sSiglec-9 significantly suppressed these gene expressions. .

Test 3: sSiglec-9 suppresses differentiation induction into osteoclasts Mouse bone marrow cells were seeded in a 24-well culture dish, stimulated with MCSF and RANKL in the presence or absence of sSiglec-9, and TRAP. ⁺ differentiated into osteoclasts. The number of TRAP ⁺ cells was assessed by immunostaining and the number of viable cells by WST-8 assay. The results are shown in FIG. Treatment with sSiglec-9 during induction dose-dependently decreased the number of TRAP ⁺ cells (FIG. 8, left). On the other hand, there was no change in WST (cell proliferation) activity (FIG. 8, right). This result suggests that sSiglec-9 does not affect osteoclast survival and suppresses the induction of osteoclast differentiation.

Mouse bone marrow cells (mBM) and RAW264.7 macrophages were stimulated with MCSF and RANKL in the presence or absence of sSiglec-9 to induce differentiation into osteoclasts. Gene expression of the osteoclast markers TRAP and cathepsin K (ctsk) was assessed using quantitative real-time PCR methods. The results are shown in FIG. sSiglec-9 treatment during induction decreased gene expression of TRAP and cstk. This result suggests that sSiglec-9 suppresses osteoclast differentiation.

Test 4: sSiglec-9 prevents osteoporosis and suppresses progression of osteoporosis model mice (OVX mice) were prepared as follows. C57BL/6J (female, 8 weeks old) was anesthetized by intraperitoneal administration of a mixed anesthesia of three types (medetomidine hydrochloride, mitasozolam, butorphanol tartrate). The body hair on the back was shaved with clippers and depilatory cream, and then fixed in a prone position. A #11 scalpel was used to make an incision of about 1 cm in the midline of the back. Using forceps, the incision was moved to the right side of the lumbar spine to confirm the subperitoneal ovary. A blunt dissection was made into the peritoneum over the perovarian fat using the tips of sharp curved forceps. The tip of sharp curved tweezers was opened by about 1 to 2 mm to grasp the fat and pulled it out from the incision. Further, the ovary, fallopian tube, and part of the uterus were pulled, and the fallopian tube side and adipose tissue side of the ovary were ligated with 4-0 polyglycolic acid absorbable sutures to remove the ovaries. Other tissues were put back into the abdominal cavity and the peritoneum was simply sutured with 4-0 polyglycolic acid absorbable thread. The skin was closed with suture clips. They were placed in a heater at 37°C and observed until they awoke from anesthesia.

After ovariectomy, 1 μg/200 ul of sSiglec-9FC was administered to the tail vein every 3 days (FIG. 10). A control group received PBS. Twenty-eight days after ovariectomy, the lower extremities were removed and photographed with a μCT imaging device (FIG. 11). A bone component region was extracted for the femur, and the following bone parameters were measured. The measurement area was set between 1 and 2.5 mm from the lower end of the growth plate of the distal femur, which is a site where remarkable changes in cancellous bone were observed by macroscopic observation. Bone extraction was performed with a cutoff value of 315 mg/cm ³ .
・Bone volume / Total volume, BV/TV, %
・Average trabecular thickness (Tb.Th, μm)
・Mean trabecular number per unit length (Trabecular number, Tb.N, 1/mm)
・ Trabecular separation (Tb.Sp, mm)

The results are shown in Fig. 12. In the sSiglec-9 group, BV/TV, Tb. Th, Tb. N was significantly improved over the control group, and Tb. An improvement tendency of Sp was observed. These results indicate that sSiglec-9 can prevent osteoporosis and slow its progression.

The present disclosure relates to treatment or prevention of bone disease, and can be used in the medical field.

Claims (11)

1. A composition for treating or preventing bone disease, comprising a secretory sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9).
2. The composition of claim 1, wherein the secreted Siglec-9 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:2 or 3.
3. The composition according to claim 1 or 2, wherein the secreted Siglec-9 comprises the amino acid sequence of SEQ ID NO: 2 or 3.
4. The composition according to any one of claims 1 to 3, wherein the bone disease is osteoporosis, osteoarthritis or femoral head necrosis.
5. The composition according to any one of claims 1 to 4, wherein the bone disease is osteoporosis.
6. The composition according to any one of claims 1 to 5 for treating or preventing osteoporosis in rheumatoid arthritis patients.
7. The composition according to any one of claims 1 to 6, further for treating or preventing rheumatoid arthritis.
8. A composition for increasing bone density, containing secreted Siglec-9.
9. A composition for preventing bone fractures containing secreted Siglec-9.
10. A composition for suppressing osteoclast differentiation, containing secretory Siglec-9.
11. A composition for suppressing the inflammatory response of fibroblasts, containing secreted Siglec-9.

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