JP6841427B2 - Treatment or prevention of malignant bone tumors with sclerostin - Google Patents

Description

Techniques for the treatment or prevention of bone tumors using sclerostin are disclosed.

Among malignant tumors, sarcomas of non-epithelial malignancies are less frequent than cancers of epithelial malignancies, and due to the large number of types, it cannot be said that pathological and etiological analysis is progressing.

Therefore, there are many tumors for which no effective treatment method has been established. Currently, treatment for malignant bone tumors is a combination of surgery, chemotherapy and radiation therapy. For osteosarcoma, which is the most common malignant bone tumor, the current multidrug therapy with anticancer drugs was started in the 1990s, and the 5-year survival rate improved from about 10% to more than 70%. .. However, there are many cases with a poor prognosis, such as cases with lung metastasis at the first visit and no surgical indication (5-year survival rate of about 50%) and cases of early recurrence after treatment (3-year survival rate of about 0%). In recent years, many new drugs such as molecular-targeted drugs have been developed for chemotherapy for other carcinomas, but they are not indicated for malignant bone tumors and their effects are limited.

Sclerostin, one of the inhibitors of the classical Wnt pathway, is a protein secreted by bone cells. It is highly specific to bone tissue and suppresses bone formation by inhibiting the classical Wnt pathway, causing osteoporosis (Non-Patent Documents 1 to 3). The anti-sclerostin antibody has already been clinically applied to the treatment of osteoporosis overseas as a bone formation promoter (Non-Patent Document 4).

van Bezooijen RL, et al. J Exp Med. 2004 Mar 15; 199 (6): 805-14. Moester M, et al. Calcif Tissue Int. 2010 Aug; 87 (2): 99-107. Weivoda MM, Curr Osteoporos Rep. 2014 McClung MR, N Engl J Med. 2014 Jan 30; 370 (5): 412-20.

Although the development of new anticancer agents targeting malignant bone tumors is eagerly desired, the current situation is that effective therapeutic agents for malignant bone tumors have not been newly developed for more than 20 years.

One object of the present invention is to treat or prevent a bone tumor by utilizing the action of sclerostin on a bone tumor.

As a result of diligent research, the present inventor has found that sclerostin has an effect of suppressing the growth of bone tumor cells. We also found that sclerostin has an effect of suppressing the migration of bone tumor cells. The present invention has been completed based on the findings and includes the following aspects.

(1) Composition item 1. A composition for the treatment or prevention of bone tumors, which comprises sclerostin.
Item 2. Item 2. The composition according to Item 1, wherein the sclerostin has the amino acid sequence of (a) below;
(A) The 28th to 210th amino acid sequences of SEQ ID NO: 1.
Item 3. Item 2. The composition according to Item 1, wherein the sclerostin has the amino acid sequence of the following (b), and the sclerostin has an inhibitory effect on bone formation;
(B) An amino acid sequence having 80% or more identity with the 28th to 210th amino acid sequences of SEQ ID NO: 1.
Item 4. Item 3. The composition according to Item 3, wherein the identity is 90% or more.
Item 5. Item 3. The composition according to Item 3, wherein the identity is 95% or more.
(B) Amino acid sequence with the 28th to 210th amino acid sequences of SEQ ID NO: 1.
Item 6. The composition according to any one of Items 3 to 5, wherein the site corresponding to the 117th asparagine of SEQ ID NO: 1 is conserved in the amino acid sequence of (b).
Item 7. The composition according to any one of Items 3 to 5, wherein the region corresponding to the 115th to 127th regions of SEQ ID NO: 1 is conserved in the amino acid sequence of (b).
Item 8. Item 8. The composition according to any one of Items 1 to 7, wherein the sclerostin is synthesized by eukaryotic cells.
Item 9. Item 8. The composition according to Item 8, wherein the sclerostin is a recombinant protein.
Item 10. Item 8. The composition according to any one of Items 1 to 9, wherein the bone tumor is a malignant bone tumor.
Item 11. Item 10. The composition according to Item 10, wherein the malignant bone tumor is a primary malignant bone tumor or a metastatic bone tumor.
Item 12. Item 2. The composition according to Item 11, wherein the primary malignant bone tumor is osteosarcoma, chondrosarcoma, myeloma, malignant lymphoma, Ewing's sarcoma, undifferentiated polymorphic bone tumor, spinal cord tumor, or the like.
Item 13. Item 2. The composition according to Item 11, wherein the metastatic bone tumor is metastatic cancer.
Item 14. Item 2. The composition according to any one of Items 1 to 13, wherein the composition is used in combination with another chemotherapeutic agent.
Item 15. Item 2. The composition according to any one of Items 1 to 14, wherein the composition is used in combination with a bone resorption inhibitor.
Item 16. Item 2. The composition according to Item 1 to Item 15, wherein the composition is used for suppressing infiltration or metastasis of a bone tumor.

(2) Treatment method Item 1. A method for treating a bone tumor, which comprises the step of administering sclerostin to an individual having a bone tumor.
Item 2. Item 2. The therapeutic method according to Item 1, wherein the sclerostin has the amino acid sequence of (a) below;
(A) The 28th to 210th amino acid sequences of SEQ ID NO: 1.
Item 3. Item 2. The therapeutic method according to Item 1, wherein the sclerostin has the amino acid sequence of the following (b), and the sclerostin has an inhibitory effect on bone formation;
(B) An amino acid sequence having 80% or more identity with the 28th to 210th amino acid sequences of SEQ ID NO: 1.
Item 4. Item 3. The treatment method according to Item 3, wherein the identity is 90% or more.
Item 5. Item 3. The treatment method according to Item 3, wherein the identity is 95% or more.
Item 6. Item 3. The therapeutic method according to Item 3, wherein the site corresponding to the 117th asparagine of SEQ ID NO: 1 is conserved in the amino acid sequence of (b).
Item 7. Item 3. The therapeutic method according to Item 3, wherein in the amino acid sequence of (b), the region corresponding to the 115th to 127th regions of SEQ ID NO: 1 is conserved.
Item 8. Item 2. The therapeutic method according to Item 1, wherein the sclerostin is synthesized by eukaryotic cells.
Item 9. Item 8. The therapeutic method according to Item 8, wherein the sclerostin is a recombinant protein.
Item 10. Item 8. The treatment method according to any one of Items 1 to 9, wherein the bone tumor is a malignant bone tumor.
Item 11. Item 10. The treatment method according to Item 10, wherein the malignant bone tumor is a primary malignant bone tumor or a metastatic bone tumor.
Item 12. Item 2. The treatment method according to Item 11, wherein the primary malignant bone tumor is osteosarcoma, chondrosarcoma, myeloma, malignant lymphoma, Ewing's sarcoma, undifferentiated polymorphic bone tumor, spinal cord tumor, or the like.
Item 13. Item 8. The treatment method according to Item 11, wherein the metastatic bone tumor is metastatic cancer.
Item 14. Item 2. The treatment method according to Item 1, further comprising a step of administering another chemotherapeutic agent.
Item 15. Item 2. The treatment method according to Item 1, further comprising a step of administering a bone resorption inhibitor.
Item 16. A method for treating a bone tumor, which comprises a step of administering sclerostin to an individual from which the bone tumor has been removed.

Bone tumor cell proliferation and / or bone tumor cell migration can be suppressed.

The number of cells measured by the alamarBlue assay is shown in a graph. The white bar indicates the control group, and the black bar indicates the sclerostin-added group. **: p <0.01. A phase-contrast microscope image of the Scratch assay is shown. For the control group and the sclerostin 100 ng / ml addition group, the state of cells 5 hours and 7 hours after the start of scratching is shown. The amount of change in the area of the gap where the cells were peeled off by the Scratch assay is shown in a graph. The white bar indicates the control group, and the black bar indicates the sclerostin-added group. *: p <0.05. A is a diagram showing an outline of the migration assay. B is a graph showing the result of the migration assay. The white bar indicates the control group, and the black bar indicates the sclerostin-added group. *: p <0.05. ^{The tumor volume (mm 3} ) in the osteosarcoma model mouse is shown graphically. The white bar indicates the control group, and the black bar indicates the sclerostin-administered group. *: p <0.05. A indicates the survival rate of osteosarcoma model mice. The control group is shown by a broken line, and the sclerostin 80 ng / g (body weight) administration group is shown by a solid line. The significant difference was p = 0.001. B is a graph showing the result of the migration assay. The white bar indicates the control group, and the black bar indicates the sclerostin-administered group. *: p <0.05.

1. 1. Composition The composition preferably contains sclerostin.
Sclerostin can be a peptide having an amino acid sequence derived from wild-type and variants of sclerostin from various animals, birds and fish. For example, sclerostin is the following (a-1), (a-2), (b-1), (b-2), (b-3), (c-1), (c-2) or (c). It is preferably the amino acid sequence of -3).

(A-1) The 28th to 210th amino acid sequences of SEQ ID NO: 1, which is the amino acid sequence of human sclerostin,
(A-2) The 24th to 210th amino acid sequences of SEQ ID NO: 1,
(B-1) The identity with the 28th to 210th amino acid sequences of SEQ ID NO: 1 is 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more. , 95% or more, 96% or more, 97% or more, 98% or more, 99% or more amino acid sequence,
(B-2) In the amino acid sequence of (b-1), the site corresponding to the 117th asparagine of SEQ ID NO: 1 is a conserved amino acid sequence.
(B-3) In the amino acid sequence of (b-1), the region corresponding to the 115th to 127th regions of SEQ ID NO: 1 is a conserved amino acid sequence.
(C-1) The 28th to 211th amino acid sequences of SEQ ID NO: 2, which is the amino acid sequence of mouse sclerostin,
(C-2) The 24th to 211th amino acid sequences of SEQ ID NO: 2,
(C-3) Amino acid sequences of sclerostins such as chimpanzees, rhesus monkeys, cattle, chickens, rats, and zebrafish (eg, The National Center for Biotechnology Information (NCBI) Reference Sequo .2, NP_001115986.1, NP_08573.1, XP_0049486088.1, XP_001340683.1, etc.), and the identity with the 28th to 210th amino acid sequences of SEQ ID NO: 1 is 80% or more, 85% or more, Amino acid sequence of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.

"Having an amino acid sequence" includes including an amino acid sequence and consisting of an amino acid sequence. When having the above-mentioned amino acid sequence, the above-mentioned (a-1), (a-2), (b-1), (b-2), (b-3), (c-) are contained in one peptide. It is preferable that the amino acid sequences of 1), (c-2) or (c-3) are continuous.

"Identity" can be calculated using commercially available analysis tools available on the market or through telecommunications lines (Internet), for example, NCBI's homology algorithm BLAST (Basic local alignment search tool) http: // www. ncbi. nlm. nih. It can be calculated by using the default (initial setting) parameters in gov / BLAST /.

"The site corresponding to the 117th asparagine of SEQ ID NO: 1 is conserved" means that the amino acid sequence of another sclerostin was analyzed when the alignment was analyzed between the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of another sclerostin. In, it means that the portion corresponding to the 117th asparagine of SEQ ID NO: 1 is the same asparagine.

"The region corresponding to the 115th to 127th regions of SEQ ID NO: 1 is conserved" means that when the alignment between the amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of another sclerostin is analyzed, the other sclerostins This means that the portion corresponding to the 115th to 127th regions of SEQ ID NO: 1 is the same amino acid sequence as the 115th to 127th regions of SEQ ID NO: 1.

Amino acids of (a-1), (a-2), (b-1), (b-2), (b-3), (c-1), (c-2) or (c-3) The sclerostin having a sequence preferably has at least one action selected from the group consisting of an osteosarcoma cell growth inhibitory action, an osteosarcoma cell migration inhibitory action, and a bone formation inhibitory action.

The presence or absence of the osteosarcoma cell growth inhibitory effect and the osteosarcoma cell migration inhibitory effect can be confirmed according to the method described in Examples described later.

The bone formation inhibitory action is the above-mentioned (a-1), (a-2), (b-1), (b-2), (b-3), (c-1), (c-2) or ( When sclerostin having the amino acid sequence of c-3) is administered to a mouse, for example, it can be determined whether or not the bone mass is reduced. Alternatively, in an in vitro experiment, the above (a-1), (a-2), (b-1), (b-2), (b-3), (c-1), (c-2) or The bone formation inhibitory effect can be confirmed using whether or not sclerostin having the amino acid sequence of (c-3) suppresses the classical Wnt pathway (Wnt / β-catenin pathway) as an index. For example, in cells expressing the receptor complex of Frizzled and Lrp5 / 6, the above (a-1), (a-2), (b-1), (b-2), (b-3), ( Western blotting, immunostaining, etc. indicate that Wnt is allowed to act on sclerostin having the amino acid sequence of c-1), (c-2) or (c-3) to suppress the accumulation of β-catenin in the cytoplasm. By evaluating with, it is possible to determine whether or not there is a bone resorption inhibitory effect. Alternatively, cells expressing the Frizzled and Lrp5 / 6 receptor complex may be subjected to the above (a-1), (a-2), (b-1), (b-2), (b-3), ( By reacting sclerostin having the amino acid sequence of c-1), (c-2) or (c-3) with Wnt, the expression of mRNA such as Axin2 and Cyclin D, which are target genes of the classical Wnt pathway, is suppressed. For example, by evaluating by real-time PCR, it is possible to determine whether or not there is a bone resorption inhibitory effect.

Sclerostin may be obtained by purifying a naturally occurring substance, or may be obtained as a recombinant peptide (genetical recombinant peptide) or a chemically synthesized peptide. As the host for producing the recombinant peptide, Escherichia coli or eukaryotic cells (yeast, insect cells, mammalian cells, avian cells, etc.) can be used, but eukaryotic cells are preferably used, and more preferably mammalian cells are used. Can be done. As a vector for producing a recombinant peptide, a known vector such as a plasmid vector or a viral vector can be used.

The recombinant peptide may contain amino acid sequences such as His tag, GST tag, FLAG tag, MAT tag, Myc tag, HA tag, immunoglobulin Fc moiety and the like.

Sclerostin may be modified with modifying groups that can be added in nature, such as sugars and lipids. Further, sclerostin may be modified with a compound such as polyethylene glycol.

Sclerostin may be used as it is as a composition, but is preferably prepared as a composition by combining an appropriate carrier or additive. The composition includes a pharmaceutical composition and a food and drink composition. As carriers and additives used in the preparation of pharmaceutical compositions, various substances commonly used in ordinary drugs depending on the dosage form of the pharmaceutical composition, such as excipients, binders, disintegrants, lubricants, etc. Examples thereof include colorants, flavoring agents, odorants, and surfactants.

When the pharmaceutical composition is orally administered, the dosage form is not particularly limited, but tablets, powders, granules, capsules (including hard capsules and soft capsules), liquids, pills, suspensions. Examples thereof include agents and emulsions. When the pharmaceutical composition is administered parenterally, examples thereof include injections, infusions, suppositories, nasal drops, and transpulmonary administrations. The pharmaceutical composition is preferably administered parenterally.

When the pharmaceutical composition is an oral solid composition such as tablets, powders, granules, rounds, capsules, etc., as a carrier, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate , Kaolin, crystalline cellulose, silicic acid, methyl cellulose, glycerin, sodium alginate, gum arabic, etc .; simple syrup, pudo sugar solution, starch solution, gelatin solution, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, carboxymethyl cellulose, cellac , Methyl cellulose, ethyl cellulose, water, ethanol, potassium phosphate, etc .; dried starch, sodium alginate, canten powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid Disintegrants such as monoglyceride, starch, lactose; disintegrators such as sucrose, stearic acid, cacao butter, hydrogenated oil; absorption enhancers such as sodium lauryl sulfate; moisturizers such as glycerin, starch; starch, lactose, kaolin, Adsorbents such as bentonite and colloidal silicic acid; purified starch, stearate, boric acid powder, lubricants such as polyethylene glycol and the like can be used. Further, the tablet may be a tablet coated with a normal skin, for example, a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, a multi-layer tablet, or the like.

When the pharmaceutical composition is an oral solid composition of pills, as a carrier, for example, excipients such as glucose, lactose, starch, cocoa butter, hardened vegetable oil, kaolin, talc; gum arabic powder, Binders such as talc powder and gelatin; disintegrants such as laminarin and canten can be used.

When the pharmaceutical composition is an oral solid composition of a capsule, the capsule is prepared by mixing sclerostin with various carriers exemplified above and filling it in a hard capsule, a soft capsule, or the like. To.

When the pharmaceutical composition is a liquid preparation, it may be an aqueous or oily suspension, a solution, a syrup, or an elixir preparation, and is prepared according to a conventional method using ordinary additives.

In the preparation when the pharmaceutical composition is an injection, as a carrier, for example, a diluent such as water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like. Acidity regulators such as sodium citrate, sodium acetate, sodium phosphate; buffers such as dipotassium phosphate, trisodium phosphate, sodium hydrogen phosphate, sodium citrate; sodium pyrosulfate, EDTA, thioglycolic acid, Stabilizers such as thiolactic acid; sugars such as mannitol, inositol, maltose, sucrose, and lactose can be used as molding agents when freeze-dried. In this case, a sufficient amount of glucose or glycerin to prepare an isotonic solution may be contained in the pharmaceutical preparation, or a usual solubilizing agent, soothing agent, local anesthetic or the like may be added. Is also good. These carriers can be added to produce subcutaneous, intramuscular, and intravenous injections by conventional methods.

When the above preparation is an intravenous drip, the administration compound can be prepared by dissolving it in an isotonic electrolyte infusion preparation based on physiological saline, Ringer's solution, or the like.

The dose of the pharmaceutical composition is not particularly limited as long as the effects of the present invention are exhibited, and may be appropriately set depending on the dosage form, the age of the patient, the sex, the degree of medical condition, etc., but is converted into, for example, the amount of sclerostin. Then, about 0.01 to 100 mg per body surface area can be administered.

Food and drink compositions include general foods and foods with health claims (foods with functional claims, foods with nutritional claims, foods for specified health use). The definition and classification of foods with health claims are as stipulated in Japan's Health Promotion Law and Food Sanitation Law.

The food and drink composition also includes food and drink (pet food) for pets (dogs, cats, hamsters, rabbits, birds, etc.) and food and drink (feed composition) for livestock (cattle, pigs, poultry). Is done.

The food and drink composition is not particularly limited, but for example, beverages (eg, milk beverages, lactic acid bacteria beverages, frozen desserts containing fruit juice, carbonated beverages, fruit juice beverages, vegetable beverages, vegetable / fruit beverages, alcoholic beverages, sports Beverages, powdered beverages, tea beverages, etc.), frozen desserts (eg jelly, bavarois, pudding, etc.), ice cream (eg ice cream, ice milk, lacto ice, sherbet), confectionery (eg cookies, biscuits, okaki, candy) , Chocolates, gums), breads, noodles (eg Chinese noodles, pasta, udon, soba, plain noodles), soups (including powdered or solid soups), seasonings (eg dressings, jellies, sauces, mayonnaise) Sama sauce, sauce) and so on.

In addition to foods and drinks having the above-mentioned forms, food and drink compositions include foods and drinks in the form of supplements, foods for children (including baby foods) and foods for the sick (foods for people requiring nursing care, and dysphagia). Includes personal foods). When prepared as a food or drink composition in the form of such a supplement, a food for children or a food for the sick, for example, a liquid (drink), a syrup, a dry syrup, or a jelly can be easily taken continuously. Preparations should be prepared in the form of preparations (including those for preparation at the time of use; the same shall apply hereinafter), granules, powders, rounds, tablets, capsules (hard capsules, soft capsules), troches, chewables, etc. Is preferable. Liquid preparations (drinks), jelly preparations, granules, tablets, capsules (hard capsules, soft capsules) are preferable, and liquid preparations (drinks) and jelly preparations are more preferable. These can be prepared according to known methods in the same manner as the above-mentioned pharmaceutical compositions.

If the national law of each country prohibits the labeling of the relationship with the disease on the food and drink composition, the relationship with the disease can be changed to a labeling format that does not conflict with the domestic law.

The inoculation amount of the food and drink composition is not particularly limited as long as the effect of the present invention is exhibited, and may be appropriately set depending on the dosage form, the age, sex, degree of medical condition, etc. of the inoculator. It is possible to inoculate about 0.01 to 100 mg per body surface area in terms of the amount of.

The composition can be used for the treatment, prevention and amelioration of bone tumors. Here, "treatment" includes suppressing the growth of a bone tumor, eliminating the bone tumor, suppressing the invasion or metastasis of the bone tumor, and / or other antitumor therapies (radiotherapy, tumor site). Includes assistance with excision, chemotherapy, etc.). "Prevention" includes preventing the development of bone tumors and / or suppressing the recurrence of bone tumors. "Amelioration" includes shrinking the bone tumor and / or reducing the infiltration or metastasis of the bone tumor.

Bone tumors include benign and malignant bone tumors. A malignant bone tumor is preferable as a disease to be administered or ingested of the composition. The malignant bone tumors include primary malignant bone tumors and metastatic bone tumors. The primary malignant bone tumors include osteosarcoma, chondrosarcoma, myeloma, malignant lymphoma, Ewing's sarcoma, undifferentiated polymorphic sarcoma of bone, spondyloma and the like. The metastatic bone tumor includes a metastatic tumor of cancer (epithelial malignant tumor) or sarcoma (non-epithelial malignant tumor). Metastatic cancer is preferably the disease to be administered or inoculated with the composition. Examples of the primary cancer of metastatic cancer include lung cancer, breast cancer, prostate cancer, kidney cancer, liver cancer, gastric cancer and the like.

The composition can be used in combination with other antitumor therapies (radiation therapy, tumor resection, chemotherapy, etc.).

The method of using the composition in combination with other antitumor therapies is not particularly limited as long as it is a mode of use that produces the effects of the present invention. For example, the composition may be used in parallel with the initiation of other antitumor therapies. Alternatively, the use of the composition may be initiated prior to the initiation of the other antitumor therapy or after the initiation of the other antitumor therapy. In this case, if the other antitumor therapy is radiation therapy or chemotherapy with another chemotherapeutic agent, the use of the composition and the other antitumor therapy may be alternated. Furthermore, if the other antitumor therapy is radiation therapy or chemotherapy using another chemotherapeutic agent, after starting the other antitumor therapy, the other antitumor is adjusted according to the degree of shrinkage of the tumor tissue. The composition may be used in combination from the middle of the therapy, or conversely, another antitumor therapy may be started from the middle of the composition after the start of use.

For example, chemotherapy is a treatment in which a chemotherapeutic agent such as an anticancer agent, a hormonal agent, or an immunostimulant is administered. Chemotherapeutic agents other than sclerostin include alkylating agents such as cyclophosphamide, iphosphamide, busulfan, melfaran, bendamstin hydrochloride, nimustin hydrochloride, lanimustin, dagalvazine, procarbazine hydrochloride temozolomid; methotrexate, pemetrexed sodium, fluorouracil. , Doxyflulysin, capecitabin, tegafur, citarabin, citarabin ocphosphate hydrate, enocitabine, gemcitabine hydrochloride, mercaptopurine hydrate, fludarabin phosphate ester, nerarabin, pentostatin, cladribine, levophorinate calcium, holinate calcium, hydroxycarbamide, Metabolic antagonists such as L-asparaginase and azacitidine; doxorubicin hydrochloride, daunorbisin hydrochloride, pirarubicin, epirubicin hydrochloride, idarubicin hydrochloride, acralbisin hydrochloride, amurubicin hydrochloride, mitoxanthron hydrochloride, mitomycin C, actinomycin D, Antitumor antibiotics such as bleomycin, pepromycin sulfate, dinostatin styramer; microvascular inhibition such as vincrystin sulfate, vinblastin sulfate, bindesin sulfate, binolerubin tartrate, paclitaxel, docetaxel hydrate, elibrine mesylate, etc. Drugs; anastrosol, exemethan, retrozol, tamoxyphen citrate, tremiphen citrate, flubestland, flutamide, bicartamide, medroxyprogesterone acetate, estramstin phosphate sodium hydrate, goseleline acetate, Hormonal agents such as leuprorelin acetate; platinum preparations such as cisplatin, milliplatin hydrate, carboplatin, nedaplatin, oxaliplatin; topoisomerase I inhibitors such as irinotecan hydrochloride hydrate, nogitecan hydrochloride; topoisomerases such as etopocid and sobzoxane II inhibitors; cytokines such as interferon gamma-1a, teseloykin, selmoloikin; antibody drugs such as tratuzumab, rituximab, gemtuzumab ozogamicin, bebashizumab, setuximab; radioimmunotherapy drugs such as ibritsumomab thiuxetane combination drug; Imatinib mecil, voltezomib, errotinib hydrochloride, sorafenibutosylate, snitinibrinate, salidomide, nirotinib hydrochloride hydrate, dasatinib hydrate, lapatinibtosilate hydrate, Molecular-targeted drugs such as everolimus, lenalidomide hydrate, dexamethasone, temsirolimus, vorinostat, tretinoin, tamibarotene; non-specific immunostimulants such as OK-432, dried BCG, kawatake polysaccharide preparation, lentinan, ubenimex; acegraton, porphy Examples thereof include marsodium, talapolphin sodium, ethanol, arsenic trioxide and the like.

These chemotherapeutic agents may be used alone or in combination. The dose can be appropriately set for each drug according to a known dose and according to the patient's condition, tumor size, and degree of progression.

Radiation therapy can be performed by irradiating the affected area with X-rays or electron beams. Here, the conditions of X-ray irradiation differ depending on the degree and size of the tumor, but usually, irradiation with a line dose of 1.5 to 3 Gy, preferably about 2 Gy is performed 2 to 5 times a week, preferably 4 to 4 to a week. A method of performing the procedure about 5 times for 1 to 5 weeks can be mentioned. The total dose may be about 20 to 80 Gy, preferably about 40 to 80 Gy, and more preferably about 50 to 70 Gy. The conditions for electron beam irradiation also differ depending on the degree and size of the tumor, but usually, irradiation with a line dose of 2 to 5 Gy, preferably about 4 Gy, is performed 1 to 5 times a week, preferably 2 to 3 times a week. The method of carrying out over 1 to 5 weeks can be mentioned. The total dose can be about 30 to 80 Gy, preferably about 40 to 70 Gy.

Sclerostin is preferably used in combination with a bone resorption inhibitor because administration may cause bone loss.

Bone resorption inhibitors include anti-RANKL monoclonal antibody drugs such as denosumab; female hormone drugs such as estriol, estrogen and estradiol; selective estrogen receptor modulators such as raloxifene and bazedoxyphene; calcitonin drugs such as elcatonin and salkecalcitonin; Examples thereof include bisphosphonate drugs such as disodium etidronate, sodium alendronate, sodium lysedronate, minodronic acid, and ibandronic acid.

These bone resorption inhibitors may be used alone or in combination. The dose can be appropriately set for each drug according to a known dose and according to the bone mass of the patient and the like.

The method of using the composition and the bone resorption inhibitor in combination is not particularly limited as long as it is a mode of use that exhibits the effects of the present invention. For example, the bone resorption inhibitor may be used in parallel at the same time as the start of use of the composition. Alternatively, the use of the bone resorption inhibitor may be started prior to the start of use of the composition or after the start of use of the composition. In this case, when the other antitumor therapy is radiotherapy or chemotherapy using another chemotherapeutic agent, the composition and the bone resorption inhibitor may be used alternately. Further, after the start of use of the composition, a bone resorption inhibitor may be used in combination from the middle of use of the composition in accordance with the change in bone mass.

2. 2. Treatment method It is preferable to use sclerostin for treatment. Preferably, it is a method for treating a bone tumor, which comprises a step of administering sclerostin to an individual having a bone tumor (preferably a human). The treatment method may further include the step of administering another chemotherapeutic agent. In addition, the treatment method may be combined with the step of administering the bone resorption inhibitor. In addition, sclerostin may be administered to individuals from whom the bone tumor has been removed by other antitumor therapies to prevent the metastasis of the bone tumor or to prevent the growth of metastasized tumor cells.

For sclerostin, the description in the composition section above is incorporated herein by reference.

Further, in the treatment method using sclerostin, sclerostin may be used as it is, or the composition may be used. For the dose of sclerostin, the description in the section of the above composition is incorporated herein by reference.

In addition, the description of the above composition section is also incorporated herein for the description of "bone tumor", "antitumor therapy", and "bone resorption inhibitor".

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not construed as being limited to Examples.
In addition, the animal experiments described in the examples were carried out in accordance with the experimental rules such as animal experiments at Shinshu University.

1. 1. Recombinant sclerostin In this example, SOST, Human, Recombinant, Carrier-free 25 μg 1406-ST-025 / CF (R & D Systems) was used as the recombinant sclerostin in the experiment using human osteosarcoma cell line HOS cells. .. The human recombinant sclerostin is a peptide from the 24th glutamine to the 213rd tyrosine of the amino acid sequence shown in NCBI registration number UniProtKB / Swiss-Prot: Q9BQB4.1 (SEQ ID NO: 1), and is 7 on the N-terminal side. It has a histag consisting of one histidine residue.

In experiments using a mouse model of osteosarcoma, SOST, Mouse, Recombinant, Carrier-free 25 μg 1589-ST-025 / CF (R & D Systems, Inc.) was used as the recombinant sclerostin. The mouse recombinant sclerostin is a peptide from the 24th glutamine to the 211th tyrosine of the amino acid sequence shown in NCBI Reference Sequence: NP_077769.4 (SEQ ID NO: 2), and consists of 7 histidine residues on the N-terminal side. Has a histag.

The human and mouse recombinant sclerostin is expressed in a mouse NS0-derived myeloma cell line.

The human recombinant sclerostin was dissolved in sterilized PBS to 200 μg / ml to prepare a stock solution. A stock solution was prepared for the mouse recombinant sclerostin in the same manner.

2. 2. Example 1: Tumor cell growth inhibitory effect of sclerostin
Using HOS cells, we investigated the effect of sclerostin on the growth of tumor cells.

(1) Method The proliferative ability of cells was evaluated by the alamarBlue assay (alamarBlue reagent: alamaeBlue DAL1100 manufactured by invitrogen). First, HOS cells were cultured in αMEM medium containing 10% FBS and antibiotics at 37 ° C. in the presence of 5% carbon dioxide. The HOS cells are divided into a control group in which HOS cells are seeded in a 10 cm dish so as to be 4 × 10 ^ 5 cells / dish and cultured in a medium containing no sclerostin, and a sclerostin-added group in which the cells are cultured in the presence of 100 ng / ml of sclerostin. Incubated for 3 days. The n number in each group was 1.

After completion of culturing, cell suspension was prepared from each dish of each group. The cell concentration was 1 × 10 ^ 4 cells / ml, and a cell suspension was prepared in a medium containing no sclerostin. Each cell suspension was dispensed into a 96-well plate at 100 μl / well at n = 8, and allowed to stand for 4 hours for cell adhesion. 10 μl of alamarBlue reagent was added to each well without removing the medium.

The fluorescence intensity of each well was measured at the time of adding the alamarBlue reagent and 1 hour after the addition of the alamarBlue reagent. For each well, the subtraction value obtained by subtracting the fluorescence intensity at the time of addition from the fluorescence intensity after 1 hour was calculated. The average and standard error of each group were calculated using the subtraction value of each well. Differences between the two groups were tested by Student's t-test.

(2) Result
The results of the alamarBlue assay are shown in FIG. The white bar indicates the control group, and the black bar indicates the sclerostin-added group. In the sclerostin-added group, the increase in the number of cells was significantly suppressed (p <0.01). From this, it was shown that sclerostin has an effect of suppressing the growth of malignant bone tumor cell lines.

3. 3. Example 2: Effect of sclerostin on tumor cell migration inhibition (1)
Using HOS cells, we investigated the effect of sclerostin on tumor cell migration.

(1) Method The migration ability of cells was evaluated by Scratch assay.
First, HOS cells were suspended in a medium so as to have a size of 4 × 10 ^ 4 / ml, and 500 μl each was dispensed into a 24-well plate. The control group was cultured in a medium containing no sclerostin, and the sclerostin-added group was cultured in the presence of 100 ng / ml of sclerostin for 3 days. The n number in each group was 3.

After completion of the culture, the medium of all wells was replaced with a medium containing no sclerostin. Then, using a pipette tip, some of the cells attached to the bottom of the well were peeled off to a certain width. After exfoliation, the medium of each well was exchanged with a medium containing no sclerostin in order to remove suspended cells including exfoliated cells, and the plate was allowed to stand at 37 ° C. in the presence of 5% carbon dioxide gas. At the start of culturing after exfoliation, 5 hours and 7 hours after the start of culturing, the exfoliated gaps were imaged under a phase-contrast microscope. The change in the area of the gap was quantified by measuring the area of the gap in the image captured by ImageJ software. For each well, a subtraction value was calculated by subtracting the area after 5 hours from the area at the start. The average and standard error of each group were calculated using the subtraction value of each well. Differences between the two groups were tested by Student's t-test.

(2) Result
FIG. 2 shows the state of the gap portion in the Scratch assay at the start of culturing after exfoliation, 5 hours and 7 hours after the start of culturing. In the visual observation of the images, the cells in the control group spread over time in the gaps after 5 hours and 7 hours as compared with the sclerostin-added group. On the other hand, in the sclerostin-added group, the cell spread was delayed as compared with the control group. FIG. 3 shows a graph in which this delay is quantitatively evaluated by the area of the gap portion. The white bar indicates the control group, and the black bar indicates the sclerostin-added group. In the sclerostin-added group, the decrease in the gap area was significantly suppressed (p <0.05). From this, it was shown that sclerostin has an effect of suppressing the migration of malignant bone tumor cell lines.

4. Example 3: Effect of sclerostin on tumor cell migration inhibition (2)
Furthermore, in order to evaluate the effect of sclerostin on the migration ability, a migration assay was performed to evaluate the effect of sclerostin.

(1) Method Similar to Example 1, HOS cells were first divided into a control group in which HOS cells were cultured in a medium containing no sclerostin and a sclerostin-added group in which cells were cultured in the presence of 100 ng / ml of sclerostin, and cultured for 3 days. The n number in each group was 1.

After completion of the culture, a migration assay was performed using a commercially available kit CytoSelectTM 96-well Cell Migration Assay (8 μm), Fluorometric (CBL). The outline of the migration assay is shown in FIG. 4A. Cell suspension (prepared in serum-free medium) was added to the inner wells of the two doubly stacked wells shown in FIG. 4A according to the protocol attached to the kit. The number of n in each group was 5. A BSA concentration gradient was created by using a 10% BSA mixed medium for the outer wells. According to the protocol attached to the kit, the number of cells migrating to the outer well after passing through the hole (8 μm in diameter) made in the bottom of the inner well after a certain period of time from the addition of the cell suspension is the fluorescence intensity. Measured in.

The fluorescence intensity of each cell in each group was determined, and the average and standard error of each group of the fluorescence intensity were calculated. Differences between the two groups were tested by Student's t-test.

(2) Result
The results of the migration assay are shown in FIG. 4B. The white bar indicates the control group, and the black bar indicates the sclerostin-added group. Migration was significantly suppressed (p <0.05) in the sclerostin-added group. From this, it was also shown in the migration assay that sclerostin has an effect of suppressing the migration of malignant bone tumor cell lines.

5. Example 4: Effect of sclerostin on osteosarcoma model mouse An osteosarcoma model mouse was prepared, and the antitumor effect of sclerostin in vivo was evaluated by administering sclerostin to this model mouse.

(1) Method i. Preparation of osteosarcoma model mouse The osteosarcoma model mouse was prepared by transplanting mouse osteosarcoma cell line LM8 cells into C3H / HeSlc mice. Mice purchased from Japan SLC Co., Ltd. at the age of 3 weeks were trained for 1 week, and LM8 cells suspended in PBS were added to the mice at the age of 4 weeks so that the number of LM8 cells was 1 × 10 ^ 6 per mouse. , Implanted subcutaneously on the left back.

ii. Administration of sclerostin Sclerostin was injected intraperitoneally into osteosarcoma model mice from the day of LM8 cell transplantation so that the target blood concentration was 1 μg / ml (10 times that of the in vitro experiment). Specifically, in the sclerostin-administered group, 80 ng of sclerostin per 1 g of mouse body weight was injected intraperitoneally once a day for 7 consecutive days. In addition, the control group was administered with the same volume of PBS as the sclerostin solution instead of sclerostin.

The mean survival time (days), tumor volume (visual, micro-CT), and the presence or absence of lung metastasis at death were examined for each mouse in the control group and the sclerostin-administered group.

The euthanasia criteria were tumor major axis of 17 mm or more, movement disorder, feeding disorder, abnormal aggression, and at least one of tumor ulceration.

The survival curve was prepared by the Kaplan-Meier method, and the difference between the two groups of the survival curve was tested by the log rank test. The difference in mean survival between the two groups was tested by Student's t-test.

(2) Result
The tumor volume of each group at 1 week after transplantation of LM8 cells is shown in FIG. The white bar indicates the control group, and the black bar indicates the sclerostin-administered group. The increase in tumor volume was significantly suppressed (p <0.05) in the sclerostin-administered group as compared with the control group. From this, it was shown that the inhibitory effect on tumor growth confirmed in vitro in Example 1 was also observed in vivo.

The survival curve is shown in FIG. 6A. The dotted line indicates the control group, and the solid line indicates the sclerostin administration group. A graph of mean survival is shown in FIG. 6B. The white bar indicates the control group, and the black bar indicates the sclerostin-administered group. The survival curve was also significantly (p = 0.001) better in the sclerostin-treated group than in the control group. The mean survival time was also significantly (p <0.05) longer in the sclerostin-treated group than in the control group.

In this example, in osteosarcoma model mice, sclerostin administration was shown to suppress the increase in tumor volume and improve the prognosis. In addition, sclerostin was thought to be effective in treating bone tumors.

Claims (8)

A composition for the treatment or prevention of bone tumors, which comprises sclerostin. The composition according to claim 1, wherein the bone tumor is a malignant bone tumor. The composition according to claim 2, wherein the malignant bone tumor is a primary malignant bone tumor or a metastatic bone tumor. The composition according to claim 3, wherein the primary malignant bone tumor is osteosarcoma, chondrosarcoma, myeloma, malignant lymphoma, Ewing's sarcoma, undifferentiated polymorphic bone tumor, or spinal cord tumor. The composition according to claim 3, wherein the metastatic bone tumor is metastatic cancer. The composition according to any one of claims 1 to 5, wherein the composition is used in combination with another chemotherapeutic agent. The composition according to any one of claims 1 to 6, wherein the composition is used in combination with a bone resorption inhibitor. The composition according to claims 1 to 7, wherein the composition is used for suppressing infiltration or metastasis of a bone tumor.