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(11) EP 3 050 573 A1

(12) EUROPEAN PATENT APPLICATION published in accordance with Art. 153(4) EPC

(43) Date of publication: (51) Int Cl.: 03.08.2016 Bulletin 2016/31 A61K 45/00 (2006.01) A61K 31/167 (2006.01) A61K 31/196 (2006.01) A61K 31/38 (2006.01) (2006.01) (2006.01) (21) Application number: 14847606.2 A61K 31/405 A61K 31/427 A61K 31/663 (2006.01) A61K 39/395 (2006.01) (2006.01) (2006.01) (22) Date of filing: 25.09.2014 A61L 27/00 A61L 31/00 A61P 19/00 (2006.01) A61P 35/00 (2006.01) G01N 33/15 (2006.01) G01N 33/50 (2006.01) G01N 33/68 (2006.01) G01N 33/92 (2006.01)

(86) International application number: PCT/JP2014/075542

(87) International publication number: WO 2015/046388 (02.04.2015 Gazette 2015/13)

(84) Designated Contracting States: (72) Inventors: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB • TAKEUCHI, Akihiko GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Kanazawa-shi PL PT RO RS SE SI SK SM TR Ishikawa 920-1164 (JP) Designated Extension States: • TSUCHIYA, Hiroyuki BA ME Kanazawa-shi Ishikawa 920-1164 (JP) (30) Priority: 25.09.2013 JP 2013199076 (74) Representative: Beckmann, Claus (71) Applicant: Nippon Chemiphar Co., Ltd. Kraus & Weisert Tokyo 101-0032 (JP) Patentanwälte PartGmbB Thomas-Wimmer-Ring 15 80539 München (DE)

(54) DRUG FOR PREVENTING, TREATING OR PREVENTING METASTASIS OF GIANT CELL TUMOR THAT OCCURS IN BONE OR SOFT PARTS, CHONDROSARCOMA, OR OSTEOSARCOMA, LOCAL INJECTION FOR ARTERIAL EMBOLIZATION, AND ARTIFICIAL BONE

(57) The present invention provides an agent for pro- phylactic treatment, therapeutic treatment, or prevention of metastasis of giant cell tumor occurring in a bone and soft tissue, chondrosarcoma, or bone sarcoma, a local infusion for artery embolization, and an artificial bone, which comprises a substance having a PPAR γ-agonistic activity and/or a PPARγ expression-inducing activity as an active ingredient. The agent for prophylactic treat- ment, therapeutic treatment, or prevention of metastasis, the local infusion for artery embolization, and the artificial bone of the present invention are a radical therapeutic agent or radical therapeutic material that can cause ap- optosis in giant cell tumor occurring in a bone and soft tissue, chondrosarcoma, or bone sarcoma to make the tumor disappear, and can induce differentiation of the tumor into fat cells to make the tumor disappear. EP 3 050 573 A1

Printed by Jouve, 75001 PARIS (FR) 1 EP 3 050 573 A1 2

Description [0005] As for occurring positions of giant cell tumor of bone (GCTB), it frequently occurs in long tubular bones, Technical Field and it occurs in a distal end of the thighbone (namely, just above the knee) or a proximal end of the tibia (name- [0001] The present invention relates to an agent for 5 ly, just below the knee) in approximately half of the total prophylactic treatment, therapeutic treatment, or preven- cases. Next to these positions, giant cell tumor of bone tion of metastasis of giant cell tumor occurring in a bone (GCTB) frequently occurs at a distal end of the radius and soft tissue, chondrosarcoma, or bone sarcoma, and (namely, the jointing position of the thumb on the radius, the like, which uses a substance having a PPARγ-ago- which is the bone on the side of the thumb among the nistic activity and/or a PPAR γ expression-inducing activ- 10 twobones connecting theelbow and the wrist), a proximal ity. The present invention also relates to a local infusion end of the humerus (namely, just under the shoulder), for artery embolization or artificial bone, which comprises and the sacrum (namely, the inverse triangle bone near a substance having a PPARγ-agonistic activity and/or a the inferior extremity of the backbone, locating at the

PPA-RY expression-inducing activity. The present inven- center of the pelvis), which are mentioned in the descend- tion further relates to a screening method for selecting a 15 ing order of occurring frequency. There are no symptoms substancethat inducesapoptosis orfat cell differentiation peculiar to giant cell tumor of bone (GCTB), and the sub- in giant cell tumor occurring in bone and soft tissue, chon- jective symptoms are such nonspecific symptoms as a drosarcoma, or bone sarcoma, as an agent for prophy- pain of affected part due to microfracture caused by re- lactic treatment, therapeutic treatment, or prevention of duction of bone strength, spontaneous pain, load-bear- metastasis of giant cell tumor occurring in bone and soft 20 ing pain, swelling, sense of heat, and difficulty in moving tissue, chondrosarcoma, or bone sarcoma. joint. [0006] Although giant cell tumor of bone (GCTB) is Background Art classified as benign tumor, it shows characteristics be- tween those of malignant and benign tumors, such as [0002] As giant cell tumors occurring in a bone and soft 25 high proliferation velocity and high recurrence rate of 10 tissue, there are known osteoclastoma occurring in a to 30%. In several percents of the cases of giant cell bone (giant cell tumor of bone, henceforth referred to as tumor of bone, it metastasizes to the lung within 1 to 10 "GCTB"), giant cell tumor of tendon sheath-localized type years, and 25% of such patients die from proliferation of occurring in a soft tissue (henceforth referred to as the tumor. Giant cell tumor of bone (GCTB) may rarely "GCTT"), pigmented villonodular synovitis (henceforth 30 convert into highly malignant sarcoma, and it is reported referred to as "PVNS"), and the like. In this specification, that, as for the prognosis of such cases, five-year survival GCTB, GCTT, and PVNS are collectively referred to as rate of such patients treated with chemotherapeutic treat- giant cell tumors. In general, among the giant cell tumors, ment and extensive resection was 50%. especially the giant cell tumor of tendon sheath (GCTT) [0007] Since giant cell tumor of bone occurring before and the pigmented villonodular synovitis (PVNS) may be 35 stop of increase of body height due to closing of the ep- generically named giant cell tumors of tendon sheath. In iphyseal plate, or giant cell tumor of bone found at an this specification, the term giant cell tumor of tendon early stage exists at the metaphysis (namely, an end part sheath refers to the giant cell tumor of tendon sheath- of diaphysis of a long tubular bone locating on the dia- localized type (GCTT) in a narrow sense, and for referring physis side with respect to the epiphysis constituting a to the giant cell tumors of tendon sheath in a broad sense, 40 part of joint), it is fundamentally considered to be a tumor the term tenosynovial giant cell tumors is used. that occurs in the metaphysis and quickly infiltrates in the [0003] As a tumor occurring in a bone and soft tissue, epiphysis. Hyperplasia of multinucleated giant cells and chondrosarcoma is also known. Similarly, as a malignant monocyte cells constitutes the major part of the patho- tumor primarily occurring in a bone tissue, bone sarcoma logical findings, and spindle cells are observed between is known. 45 them. The morphology and functions of the multinucle- [0004] Giant cell tumor of bone (GCTB) is a benign ated giant cells are similar to those of osteoclasts. It is tumor frequently occurring in circumferences of the knee thought that the body of the tumor of giant cell tumor of joints of young to middle- or advanced-aged persons, bone consists of spindle cells similar to fibroblasts and and accounts for 3 to 8% of the primitive bone tumors, osteoblastsexisting in the stroma, and the multinucleated and 15 to 20% of benign bone tumors. The morbidity of 50 giant cells and monocyte cells are cells gathering in re- the giant cell tumor of bone (GCTB) is slightly higher in sponse to a cytokine produced by the tumor cells. Al- women, and patients’ man-and-woman ratio is 1:1.3 to though it is considered that giant cell tumor of bone is 1.5. Giant cell tumor of bone (GCTB) frequently occurs probably a tumor originating in undifferentiated mesen- in 20 to 45 years old persons, and it is said that about chymal cells in the bone marrow, cell origin thereof is 150 persons newly develop this disease in every year in 55 unknown. Japan. Giant cell tumor of bone (GCTB) accounts for [0008] There is only ablative operation as the radical about 5% of all bone tumors, and about 20% of benign therapy of giant cell tumor of bone (GCTB). In usual ab- bone tumors. lative operations, phenol treatment, alcohol treatment,

2 3 EP 3 050 573 A1 4 zinc chloride treatment, freezing with liquid nitrogen, and toms of giant cell tumor of tendon sheath (GCTT), and it thermotherapeutic treatment using heat of polymeriza- occurs as hypodermic tumor in fingers with no substantial tion of methyl methacrylate bone cement are performed pain, shows slow proliferation, and generally passes sev- after curettage or excision of the lesion, for the purpose eral years until it is diagnosed by a medical examination of annihilating remaining tumor cells to prevent recur- 5 of a medical practitioner. rence, and the recurrence rate of 30 to 50% observed [0013] As the pathology of giant cell tumor of tendon without such treatments as mentioned above is thereby sheath (GCTT), orbicular-ovate to spindle-shaped histi- successfully lowered to 10 to 25%. ocyte-like monocytes showing diffusible proliferation and [0009] Although it is rare for giant cell tumor of bone osteoclast-like multinucleated giant cells are intermin- (GCTB) to follow a fatal process, it is a disease of which 10 gled in tumors showing clear borders. The cause of giant repetition of recurrence gradually spoils motor functions cell tumor of tendon sheath (GCTT) is unknown, and the of bones and joints, and at the same time, for which sur- origin of tumor cells is also unknown. gical operations cause nerve damages to greatly de- [0014] Giant cell tumor of tendon sheath (GCTT) is grades the quality of life, and therefore there is desired classified as benign tumor, and it seldom follows fatal a therapeutic treatment that is not based on surgical op- 15 process. However, it strongly tends to proliferate and eration, but on an internal medical therapeutic treatment, spread over surroundings, and it recurs in 20 to 30% of and does not invite recurrence or metastases to the lung. the cases even if extirpation operations are conducted. [0010] As for a therapeutic treatment for giant cell tu- Since giant cell tumor of tendon sheath (GCTT) strongly mor of bone (GCTB) not based on surgical operation, adheres to tendon sheath, it is hard to excise it, and if an there is investigated application to giant cell tumor of20 extirpation operation is conducted, adhesion of extensor bone of denosumab (trade name, Ranmark), which com- tendon or flexor tendon, and injury to nerves or blood prises an anti-RANKL human monoclonal antibody, and vessels are easily caused. Moreover, advance of giant is clinically used as a therapeutic agent for bone diseases cell tumor of tendon sheath (GCTT) destroys bones, caused by multiple myeloma and bone diseases caused joints, and ligaments. Therefore, there is desired a ther- by solid carcinoma metastases. However, said medica- 25 apeutic treatment of giant cell tumor of tendon sheath ment is a therapeutic agent aiming at suppressing the (GCTT) that is not based on surgical operation, but is functions of the RANKL protein required for formation based on an internal medical therapeutic treatment, and and activation of osteoclasts to prevent bone destruction, does not invite recurrence and metastasis to lung. There and is not a therapeutic agent aiming at suppressing tu- is also desired a therapeutic agent for giant cell tumor of mor proliferation itself, and as for the administration30 tendon sheath (GCTT) that can be orally administered, route, it should be subcutaneously administered. and can suppress tumor proliferation. [0011] Therefore, there is desired a therapeutic agent [0015] Pigmented villonodular synovitis (PVNS) is a for giant cell tumor of bone (GCTB) that can be orally benign tumor that occurs in relatively young adults not administered, and can suppress tumor proliferation, per olderthan 40,and slightly more frequently occurs inwom- se. 35 en. As for occurring position of PVNS, it most frequently [0012] Giant cell tumor of tendon sheath (GCTT) is a occurs in the knee joint, and also frequently occurs in the soft tissue tumor that frequently occurs around joints and large joints such as hip, leg, elbow, and shoulder joints, tendon sheaths at peripheries of arms and legs, and in and surroundings thereof. PVNS is a disease that shows particular, cases thereof developing the tumor adjacently abnormal proliferation of tissues of the synovial mem- to the tendon sheaths around interphalangeal joints of 40 brane covering the inside of joints, forms tumors, and fingers are overwhelmingly frequently observed. Giant repeatedly causes hemorrhage. It is classified as that of cell tumor of tendon sheath (GCTT) occurs around joints "diffuse type" in which tumors are densely formed like of fingers or on flexor tendons in about 85% of the cases, piles of carpet on the whole surface, and "limited type and it next frequently occurs in foot. Giant cell tumor of (nodule type)" in which tumors are serially formed in a tendon sheath (GCTT) may infiltrate in bones. Although 45 row, and the both types may simultaneously seen in not giant cell tumor of tendon sheath (GCTT) is synonymous a few cases. with nodular tenosynovitis, but it is not an inflammatory [0016] The subjective symptoms thereof include swell- disease, but it is a tumor. Although there is not known ing and dull pain of joints, sticking sense of joints, disa- any report describing exact occurrence frequency of gi- bility for bending and extending joints beyond a certain ant cell tumor of tendon sheath (GCTT), it is a disease 50 extent, hot sensation at the knee, and the like, and blood of which number of cases is next to those of lipoma and often accumulates in joints. neurilemmoma among benign soft tissue tumors that are [0017] PVNS is defined to be the same as the diffuse excised by orthopedists for the purpose of therapeutic type giant cell tumor according to the WHO classification, treatment, and it is not a rare disease. Giant cell tumors and classified as a benign soft tissue tumor. In fact, tu- of tendon sheath (GCTT) frequently occurs in adults in 55 mors of PVNS themselves do not metastasize to other their thirties to fifties, and especially frequently occurs in organs, and grow slowly, and therefore it is not a fatal women, and the patients’ man-and-woman ratio was re- disease. However, if PVNS is neglected, destruction and ported to be 1:2. There are no special subjective symp- deformation of bones advance to cause gonarthrosis and

3 5 EP 3 050 573 A1 6 greatly spoil the quality of life of patients, and therefore ated giant cartilage cells, and the like. Although the bor- it is a disease for which an early treatment is required. ders of tumors of chondrosarcoma are comparatively Moreover, since PVNS infiltrates in a diffusive manner, clear, there is observed infiltration thereof between bone completeexcision thereof is difficult,and it recursin about trabeculae (namely, finely loosen part of the spongin tis- 50% of patients. As for histological characteristics, it5 sues at the epiphysis of a long tubular bone), or in the shows diffusive proliferation of synovial cell-like mono- Havers canal (namely, passage of blood vessels locating cytes, and there are intermingled osteoclast-like multi- at the center of bone lamella system of the compact bone nucleated giant cells, foam cells, siderophores, inflam- in the diaphysis of a long tubular bone). The cause and matory cells, and the like. The cell origin of PVNS is un- cell origin of chondrosarcoma are unknown. known. 10 [0024] As the radical therapy of chondrosarcoma, [0018] There is only ablative operation as the thera- there is only ablative operation. Against chondrosarco- peutic treatment of PVNS. In ablative operation of PVNS, ma, effect of radiotherapy or anticancer drug treatment grown synovial membrane should be excised under ob- is not sufficient. Since chondrosarcoma is a malignant servation with an endoscope or after incision of joint. tumor, extensive excision is performed so as to excise However, even if the surgical operation is conducted so 15 the tumor and surrounding tissues together with covering that neither the joint capsules nor the ligamentum tissues normal tissues. Therefore, the surgical operation in- should be injured, if it infiltrates to a bone, it should also volves a risk of spoiling motor functions, and amputation be eliminated by curettage, and depending on degree of of diseased limb is sometimes unavoidable. Although the infiltration to a bone, use of an artificial joint or amputation five-year survival rate of chondrosarcoma is 70 to 80%, of leg or arm may be required. 20 there are not a few cases where a long-term process of [0019] Therefore, there is desired a therapeutic treat- ten years or longer including repetitive recurrences re- ment that is not based on surgical operation, but is based sults in death. on an internal medical therapeutic treatment. There is [0025] Therefore, there is desired a therapeutic treat- also desired a therapeutic agent for PVNS that can be ment of chondrosarcoma that is not based on surgical orally administered, and can suppress tumor prolifera- 25 operation, but is based on an internal medical therapeutic tion. treatment. There is also desired a therapeutic agent for [0020] As described above, for all of giant cell tumor chondrosarcoma that can be orally administered, and of bone, giant cell tumor of tendon sheath, and PVNS, can suppress the tumor proliferation. any effective therapies have not been developed at [0026] Bone sarcoma is a malignant tumor that prima- present, except for surgical operation. 30 rily develops in a bone tissue, and is a malignant tumor [0021] Chondrosarcoma accounts for about 20% of that directly produces osteoid (constituent element of primary malignant bone tumors, and is a malignant tumor bone tissues consisting of matrix and fibers), or bone. showing the secondly highest occurrence frequency fol- Bone sarcoma is the most frequently occurring tumor lowing that of bone sarcoma. It occurs in persons of such among the malignant tumors that primarily develop in a a broad age range as twenties to sixties, and it is com- 35 bone, and it occurs in 1 to 2 persons out of one million paratively frequently observed in, in particular, persons persons, and occurs in about 200 persons every year in of middle or advanced age of 40 years old or older, and Japan. Bone sarcoma patients in their teens account for such cases account for almost half of the total cases. As 60%, and those in their twenties account for 15% of the for the patient’s man-to-woman ratio, chondrosarcoma total bone sarcoma patients. As for the patients’ man-to- occurs about twice frequently in men compared with40 woman ratio, it slightly more frequently occurs in men women. compared with women. Bone sarcoma cases account for [0022] As for onset position of chondrosarcoma, it fre- about 33% of the total cases of malignant tumors that quentlyoccurs inthe thighbone (namely, the bone ofthigh primarily develop in bones. above knee), humerus (namely, the bone extending from [0027] Bone sarcoma frequently occurs in a long tubu- elbow to shoulder), pelvis, rib, scapula, and flat bone, 45 lar bone, and the most frequently occurring positions are, and the cases occurring in these positions account for from the highest frequency, the knee (distal position of 70 to 80% of the total cases. the thighbone (namely, position above the knee) and [0023] As for the subjective symptoms of chondrosar- proximal position of the tibia (namely, position below the coma, patients frequently notice the disease from swell- knee), of which cases account for 60%, hip joint, of which ing of affected part or dyskinesia, patients may also notice 50 cases account for 15%, shoulder joint, of which cases the disease from hard tumor giving weak pain, and pain account for 10%, and jaw, of which cases account for caused by bone fracture. Histological images of chond- 6%, and these frequencies are similar to the occurrence rosarcoma show abundant glasslike cartilage matrices frequencies of the giant cell tumor of bone for such po- or mucous matrices, and they proliferate in a lobulating sitions. As a subjective symptom of bone sarcoma, con- shape. Histological images of chondrosarcoma generally 55 tinuous pain arises, and such pain occurs in connection present (1) abundant cell components, (2) hypertrophy with sporting activities. Therefore, it may be mistaken for of nuclei, (3) appearance of hypertrophied binucleated muscular pain, and thus cautions are required. cells, (4) appearance of chromatin-abundant multinucle- [0028] Although the five-year survival rate of bone sar-

4 7 EP 3 050 573 A1 8 coma depends on medical facilities, there is a report that is also desired a therapeutic agent for bone sarcoma that it was about 70% for the cases without metastasis. Bone can suppress metastasis of bone sarcoma cells to the sarcoma frequently metastasizes to the lung, like giant lung. cell tumor of bone. Prognosis of the cases for which me- [0033] As described above, giant cell tumor of bone tastasis is found at the time of the first medical examina- 5 (GCTB), giant cell tumor of tendon sheath (GCTT), pig- tion is bad, and the five-year survival rate for such cases mented villonodular synovitis (PVNS), chondrosarcoma, is about 20% even in advanced facilities. and bone sarcoma are tumors that occur in bone soft [0029] In the histopathological sense, bone sarcoma tissues, and they are commonly characterized in that is a low differentiation spindle-shaped multinucleated there are no radical therapy other than ablative operation, sarcoma. However, histological images thereof show a 10 and even if ablative operation is conducted, repetitive wide variety of aspects, including from those apparently recurrence continuously degrades the quality of life of seen only as reactive osteogenesis or fibrosis to those patients. However, the causes and original cells thereof showing consolidation with marked bone and osteoid for- are unknown. Although histologically osteoclast-like mation, those showing marked chondrogenesis, as well multinucleated giant cells are commonly observed in gi- as those showing characteristics of utterly undifferenti- 15 ant cell tumor of bone (GCTB), giant cell tumor of tendon ated sarcoma hardly accompanied by osteoid, and thus sheath (GCTT), PVNS, and bone sarcoma, it is estimated considerably change, and it always appears in different that the body of the tumor is not the osteoclast-like multi- ways depending on positions even in the same tumor. nucleated giant cells, and therefore there are of course Appearance of multinucleated giant cells almost always not known any therapeutic treatment based on a mech- observed, and they show various appearances both in 20 anism commonly applicable to giant cell tumor of bone amount and quality, including those of osteoclast-like (GCTB), giant cell tumor of tendon sheath (GCTT), pig- gentle type to those of strange appearance apparently mented villonodular synovitis (PVNS), and chondrosar- seen to be malignant, and may sometimes show giant coma, and any attempts for developing medicaments for cell tumor of bone-like images. such therapeutic treatment. [0030] Astherapies of bone sarcoma, chemotherapeu- 25 [0034] As report concerning therapeutic agent for giant tic treatment and surgical operation are mainly conduct- cell tumor occurring in bones and soft tissues known so ed. The chemotherapeutic treatment for bone sarcoma far, there is only a report that mizoribine, which is an is characterized in that the chemotherapeutic treatment immunosuppressant based on inhibitory action against is performed in advance of surgical operation, unlike thebiosynthesis system of purine ofnucleic acids, slightly those for the other types of cancers. Already existing in- 30 inhibited proliferation of PVNS in vitro (Patent document visible micrometastases (lung, liver, bone, and the like) 1), except for the reports of the inventors of the present can be thereby eradicated or suppressed, and an effec- invention themselves. The reports of the inventors of the tive anticancer agent can also be thereby determined. present invention, to which the provisions of the excep- Therefore, it enables selection of anticancer agent to be tion to loss of novelty or grace period shall be applied, used in postoperative chemotherapeutic treatment (per- 35 are not mentioned in this section as prior art references. formed after surgical operation), or chemotherapeutic [0035] The peroxisome proliferator-activated receptor treatment to be performed at the time of recurrence. At y (PPARγ) is a transcriptional factor protein belonging to present, cisplatin, adriamycin, ifosfamide, cyclophos- the intranuclear receptor superfamily, which exists in fat phamide, ethoposide, methotrexate, doxorubicin, bleo- cells, macrophages, and the like. PPAR Y exists as a het- mycin, and caffeine are frequently used in an appropriate 40 ero-complex formed together with the retinoid X receptor combination thereof. For example, in the caffeine-com- (RXR) protein. bined therapy, cisplatin, adriamycin, and caffeine are [0036] It is considered that, in the absence of a PPAR Y used in combination. Most of these anticancer agents agonist, the hetero-complex of PPARY and RXR binds should be administered by drip infusion. with a corepressor protein complex, and binds to the [0031] The most important in topical treatment of bone 45 PPAR response element (PPRE) existing in a promoter sarcoma is to surely excise the primary lesion. However, region of a lipid metabolism-related gene in a genome considering the frequent occurrence in young persons, gene to suppress transcription of mRNAs of the genes preservation of motor functions is also important. The existing downstream thereof, including apoptosis-related number of cases not requiring amputation of leg or arm, genes, various lipid metabolism-related genes or fat cell and allowing preservation of the diseased limb is increas- 50 differentiation-related genes such as those for lipoprotein ing in recent years. However, if an artificial joint must be lipase (LPL) and fatty acid transport protein (FATP), ar- used,lifetime of artificialjoint is about 20 years, and there- teriosclerosis-related genes, and anti-inflammation-re- fore there arises a problem that, even if the disease is lated genes. It is also considered that, however, if a completely cured, a resurgical operation is required for PPARγ agonist binds to PPA-RY, a co-activator protein 55 replacing the artificial joint. complex binds to the hetero-complex of PPAR Y and RXR [0032] Therefore, there is desired a therapeutic agent in place of release of the co-repressor protein complex for bone sarcoma that can be orally administered, and from the hetero-complex of PPAR Y and RXR to promote can suppress proliferation of bone sarcoma cells. There transcription of mRNAs of the genes existing down-

5 9 EP 3 050 573 A1 10 stream from the PPAR response element (PPRE), in- document 6), and malignant melanoma (Non-patent doc- cluding the apoptosis-related genes, various lipid metab- ument 7). It is also known that thymoquinone having an- olism-related genes or fat cell differentiation-related ticancer activity increases the PPARY activity in breast genes such as those for lipoprotein lipase (LPL) and fatty cancer cells (Non-patent document 8). acid transport protein (FATP), arteriosclerosis-related 5 [0043] Besides the above, Japanese Patent Unexam- genes, and anti-inflammation-related genes. ined Publication (Kokai) No. 2005-200419 (Patent doc- [0037] As PPARγ agonists, there are known angi- ument 2) describes, in the claims, a method for thera- otensin II receptor antagonists such as irbesartan and peutic treatment of cancer using a mevalonate pathway telmisartan, thiazolidinedione derivatives, non-steroidal inhibitor and a PPAR Y agonist in combination, but it does anti-inflammatory agents, and endogenous ligands such 10 not mention any example at all. Therefore, it is an unver- as 15-deoxy-A12,14-prostagladin J2 (15d-PGJ2), 15-hy- ified invention, and does not have any meaning as a prior droxyeicosatetraenoic acid (15-HETE), 9-hydroxyocta- art. Similarly, Japanese Patent Unexamined Publication decadienoic acid (9-HODE), 13-hydroxyoctadecadieno- (Kohyo) No. 2009-533467 (Patent document 3) de- ic acid (15-HODE), nitrolinoleic acid, oxidized LDL, long scribes, in claims 9, and 14 to 17, uses of compounds chain fatty acids, eicosanoids, and lysophospholipids. 15 having the thiazolidinedione structure represented by a Long chain fatty acid refers to an aliphatic acid containing general formula for therapeutic treatment of carcinoma, 11 or more carbon atoms in the molecule. sarcoma, and giant cell tumor of bone. However, like Pat- [0038] The angiotensin II receptor antagonists are ent document 2, it does not mention any example indi- agents for lowering blood pressure by inhibiting binding cating the pharmacological activity at all. of angiotensin II, which is a pressor substance, with the 20 [0044] As described above, there are not known any receptor thereof. Irbesartan and telmisartan are angi- prior art references describing that thiazolidinedione de- otensin II receptor antagonists currently used on clinical rivatives can be used for prophylactic and therapeutic sites, and they are known to have a partial agonistic ac- treatments, or prevention of metastasis of giant cell tumor tivity for PPARγ (partial agonist), in addition to the angi- occurring in bones and soft tissues with scientific evi- otensin II receptor antagonist activity. 25 dences, except for the reports of the inventors of the [0039] There is not known any prior art reference de- present invention themselves. scribing that an angiotensin II receptor antagonist can be [0045] The non-steroidal anti-inflammatory agents are used for prophylactic or therapeutic treatment, or preven- non-steroidal agents having anti-inflammatory activity, tion of metastasis of giant cell tumor occurring in bones analgesic action, and antipyretic action, of which typical and soft tissues, chondrosarcoma, or bone sarcoma. 30 example is aspirin (namely, acetylsalicylic acid), and they [0040] The thiazolidinedione derivatives are known as are known as relatively safe drugs. It is known that, as therapeutic agents for type II diabetes mellitus, and pi- for the action mechanism of the non-steroidal anti-inflam- oglitazone and rosiglitazone are clinically used. The thi- matory agents, they provides the anti-inflammatory ac- azolidinedione derivatives exhibit activities for improving tivity, analgesic action, and antipyretic action through in- insulin resistance, suppressing saccharide production in 35 hibitory activity against cyclooxygenase 1 and/or cy- the liver, promoting saccharide incorporation in periph- clooxygenase 2. eral cells, combusting fatty acids, promoting sensitivity [0046] As the non-steroidal anti-inflammatory agents, of insulin receptor, suppressing arteriosclerosis, anti-in- there are known zaltoprofen, diclofenac, indomethacin, flammation, suppressing myocardial hypertrophy, and proglumetacin, indometacin farnesil, celecoxib, etodol- the like by eliminating hypertrophied fat cells that secrete 40 ac, meloxicam, mofezolac, acemetacin, oxaprozin, TNFα, resistin, MCP-1, PAI-1, and the like by apoptosis, acetaminophen, lornoxicam, ampiroxicam, piroxicam, or deriving them to differentiate into small fat cells that naproxen, loxoprofen, rofecoxib, ethenzamide, diflunis- secrete adiponectin into blood. al, aluminoprofen, nabumetone, ketoprofen, acetylsali- [0041] As the thiazolidinedione derivatives, there are cylic acid, ibuprofen, pranoprofen, sulindac, and the like, known pioglitazone, rosiglitazone, troglitazone, isaglita- 45 and various kinds of drugs are widely used on clinical zone, netoglitazone, rivoglitazone, balaglitazone, lobeg- sites as drugs for ameliorating such symptoms as head- litazone, englitazone, ciglitazone, and the like. However, ache, toothache, menstrual pain, and pyrexia. since they may cause critical adverse drug reaction, [0047] Proglumetacin and maleate thereof are prod- those clinically used at present as therapeutic agent for rugs that are metabolized into indomethacin in the body type II diabetes mellitus are only pioglitazone and rosigl- 50 and exhibit the efficacy. Similarly, indometacin farnesil itazone. is a prodrug that is metabolized into indomethacin in the [0042] In addition to the activity as a therapeutic agent body and exhibits the efficacy. Similarly, ampiroxicam is for type II diabetes mellitus, the thiazolidinedione deriv- a prodrug that is metabolized into piroxicam in the body atives such as pioglitazone are known on a laboratory and exhibits the efficacy. level to inhibit proliferations of colon cancer (Non-patent 55 [0048] As for use of non-steroidal anti-inflammatory documents 1 and 2), gastric cancer (Non-patent docu- agents for oncotherapy, Japanese Patent Unexamined ments 3 and 4), non-small cell type lung cancer cells Publication (Kokai) No. 2005-343802 (Patent document (Non-patent document 5), chondrosarcoma (Non-patent 4) describes in the examples that when ketoprofen was

6 11 EP 3 050 573 A1 12 transdermally administered to nude mice transplanted SS, which is a chondrosarcoma cell line, to suppress with the OST cells, which are human bone sarcoma-de- proliferation thereof, and promoted expression of PPARy rived cultured cells, the tumor weight decreased to 48% (Non-patent documents 18 and 19). of that observed in a placebo group after four weeks. [0055] Non-patent documents 11 to 19 mentioned Further, Japanese Patent Unexamined Publication (Ko- 5 above, which are reports of the inventors of the present hyo) No. 2006-501136 (Patent document 5) describes invention themselves, do not constitute prior arts of the methods for therapeutic treatments of pain, inflamma- present invention, since provisions of the exception to tion, cancer, Alzheimer’s disease, and cardiovascular loss of novelty or grace period shall be applied to them. disease using a PPARγ agonist or selective inhibitor for [0056] As described above, except for the reports of cyclooxygenase 2 in the claims thereof. However, it does 10 the inventors of the present invention themselves, there not describe at all any results of examples demonstrating are not known any prior art references describing that the pharmacological actions. non-steroidal anti-inflammatory agents can be used for [0049] As for prior art describing relation between prophylactic treatment, therapeutic treatment, or preven-

PPARY agonist and non-steroidal anti-inflammatory tion of metastasis of giant cell tumor occurring in bones agent, it was reported that diclofenac or celecoxib pre- 15 and soft tissues, chondrosarcoma, or bone sarcoma. vented occurrence of colon cancer induced by repetitive [0057] Moreover, since it is not considered that there administration of 1,2-dimethylhydrazine dihydrochloride is any radical therapy for giant cell tumor occurring in to rats, and at the same time, these agents reduced bones and soft tissues except for ablative operation, and amount of NF-kB protein in the large intestine, and in- it is considered that the first priority of the treatment of creased amount of the PPARγ protein (Non-patent doc- 20 chondrosarcoma or bone sarcoma is given to raising sur- ument 9). vival rate by extensive excision, an object of improving [0050] As for action on synovial cells derived from ability to carry out everyday activities of patients suffering rheumatism patients, it was reported that troglitazone, from giant cell tumor occurring in bones and soft tissues, indomethacin, diclofenac, oxaprozin, and zaltoprofen ac- chondrosarcoma, or bone sarcoma is not recognized. tivated PPARγ, and caused apoptosis to suppress pro- 25 liferation of the cells, but NS-398, which is a selective Prior art references cyclooxygenase 2 inhibitor, did not activate PPARγ and did not cause apoptosis, although it suppressed prolifer- Patent documents ation of the cells, and ketoprofen and acetaminophen did not cause activation of PPARγ, apoptosis, and suppres- 30 [0058] sion of the proliferation (Non-patent document 10). [0051] Furthermore, it was reported by the inventors Patent document 1: WO2008/026729 of the present invention that when zaltoprofen was al- Patent document 2: Japanese Patent Unexamined lowed to act on giant cell tumor of bone (GCTB) derived Publication (Kokai) No. 2005-200419 from a patient, it suppressed proliferation of the cells of 35 Patent document 3: Japanese Patent Unexamined the giant cell tumor of bone, promoted expression of Publication (Kohyo) No. 2009-533467 PPARy in the cells of giant cell tumor of bone, and in- Patent document 4: Japanese Patent Unexamined duced differentiation of the cells into fat cells (Non-patent Publication (Kokai) No. 2005-343802 documents 11 and 12). Patent document 5: Japanese Patent Unexamined [0052] Similarly, it was reported by the inventors of the 40 Publication (Kohyo) No. 2006-501136 present invention that when zaltoprofen or troglitazone was allowed to act on giant cell tumor of bone (GCTB) Non-patent documents derived from a patient, these drugs suppressed prolifer- ation of the cells of the giant cell tumor of bone, and pro- [0059] moted expression of PPAR γ in the cells of giant cell tumor 45 of bone, and zaltoprofen induced differentiation of the Non-patent document 1: Jpn. J. Cancer Res., cells into fat cells (Non-patent documents 13, 14, 15, and 1999;90:75-80 16). Non-patent document 2: Cancer Lett., [0053] It was also reported by the inventors of the 2010;297:65-74 present invention that in a patient who had taken zalto- 50 Non-patent document 3: FEBS Lett., profen for four weeks at a dose of 240 mg per of day, 1999;455:135-139 which is the standard dose mentioned in the package Non-patent document 4: Zhongguo yishi zazhi insert, giant cell tumor of bone disappeared, but instead, 2010;12(6):743-747 fatcell-like cellsshowing enhanced expression of PPARy Non-patent document 5: Mol. Pharmacol., were observed (Non-patent documents 13 and 17). 55 2007;72:674-685 [0054] Similarly, it was also reported by the inventors Non-patent document 6: British Journal of Cancer, of the present invention that zaltoprofen, pioglitazone, 2002; 86:1303-1309 andtroglitazone induced apoptosis in the cellsof H-EMC- Non-patent document 7: Medical Oncology,

7 13 EP 3 050 573 A1 14

2006;23(3):393-402 chondrosarcoma, or bone sarcoma to improve quality of Non-patent document 8: Biochem. Pharmacol., life of the patient. 2011;82:464-475 [0062] A still further object of the present invention is Non-patent document 9: Tumor Biology, 2010; to provide a local infusion for artery embolization and/or 31:427-436 5 artificial bone that can cure giant cell tumor occurring in Non-patent document 10: J. Pharmacol. Exp. Ther., a bone and soft tissue, chondrosarcoma, or bone sarco- 2002;302:18-25 ma, and can suppress recurrence and/or metastasis of Non-patent document 11: The Journal of the Japa- them. A still further object of the present invention is to nese Orthopedic provide Association, a method for screening for an agent for prophy- 2012;86(8):S1319:2-9-18 10 lactic treatment, therapeutic treatment, or prevention of Non-patent document 12: The Journal of the Japa- metastasis of giant cell tumor occurring in a bone and nese Orthopedic Association, 2013;87(8):1-8-22 soft tissue, chondrosarcoma, or bone sarcoma. Non-patent document 13: 2013 AAOS (American Association of Orthopedic Surgeons) Annual Meet- Means for Achieving the Object ing Abstract Paper 341 15 Non-patent document 14: 26th Eur. Musculoskeletal [0063] The inventors of the present invention, who are Oncology Society Meeting 2013, Abstract P4:103 orthopedic surgeons, incidentally encountered, at the Non-patent document 15: The Journal of the Japa- time of ablative operation of giant cell tumor of bone nese Orthopedic Association, 2013;87(6):1-2-FP3-8 (GCTB), which is the sole radical therapy of GCTB, a Non-patent document 16: ISOLS 2013 Abstract N° 20 case where cells characteristic to bone giant cells did not 205 (Annual Meeting of International Society of Limb exist in the excised bone tissues, but instead, fat cell-like Salvage 2013, Abstract N° 310) cells existed in them. While it was hardly surprising to Non-patent document 17: Anticancer Research, overlook the reason for the spontaneous recovery as a 2013;33:2169-2174 unique case, the inventors of the present invention care- Non-patent document 18: The Journal of the Japa- 25 fully investigated the pathological and therapeutic history nese Orthopedic Association, 2013;87(8):1-8-20 of the patient and heard symptoms and life conditions Non-patent document 19: ISOLS 2013 Abstract N° from the patient himself as faithfully adhering to the ba- 205 (Annual Meeting of International Society of Limb sics. During this process, the inventors of the present Salvage 2013, Abstract N° 205) invention noted the fact that the patient had taken zalto- 30 profen over four weeks at a dose of 240 mg per day, Summary of the Invention which is the standard dose indicated in the package in- sert, in order to ameliorate pain of joints caused by giant Object to be Achieved by the Invention cell tumor of bone, hit an idea that the fact had somehow related to the spontaneous recovery, and got a concept [0060] An object of the present invention is to provide 35 of the present invention that non-steroidal anti-inflamma- a method for prophylactic treatment, therapeutic treat- tory and sedative agents might suppress proliferation of ment, or prevention of metastasis of giant cell tumor oc- giant cell tumor occurring in a bone and soft tissue, chon- curring in a bone and soft tissue, chondrosarcoma, or drosarcoma, or bone sarcoma, and induce it to differen- bone sarcoma, which is not based on surgical operation. tiate into fat cells. Another object of the present invention is to provide an 40 [0064] Then, the inventors of the present invention agent for prophylactic treatment, therapeutic treatment, contacted various non-steroidal anti-inflammatory and or prevention of metastasis of giant cell tumor occurring sedative agents to cells excised from patients suffering in a bone and soft tissue, chondrosarcoma, or bone sar- from giant cell tumor occurring in a bone and soft tissue, coma, which can be orally administered, and can sup- chondrosarcoma, or bone sarcoma, and confirmed that press proliferation of tumor, per se. A further object of 45 theagents inhibited proliferationthereof, induced expres- the present invention is to provide an agent for prophy- sion of PPARY, and induced the tumor to differentiate lactic treatment, therapeutic treatment, or prevention of into fat cell-like cells. They further confirmed that PPAR Y metastasis of giant cell tumor occurring in a bone and agonists other than non-steroidal anti-inflammatory and soft tissue, chondrosarcoma, or bone sarcoma, which sedative agents also inhibited proliferation of giant cell can be orally administered, and can eliminate the tumor 50 tumor of bone (GCTB), giant cell tumor of tendon sheath and/or induce the tumor to differentiate into fat cells. (GCTT), PVNS, and chondrosarcoma, and came to ac- [0061] A still further object of the present invention is complish the present invention. to provide a medicament that can suppress recurrence [0065] The present invention thus provides the follow- and/or metastasis of giant cell tumor occurring in a bone ings. and soft tissue, chondrosarcoma, or bone sarcoma. A 55 still further object of the present invention is to provide a [1] An agent for prophylactic treatment, therapeutic medicament that can recover motor functions of a patient treatment, or prevention of metastasis of giant cell of giant cell tumor occurring in a bone and soft tissue, tumor occurring in a bone and soft tissue, chondro-

8 15 EP 3 050 573 A1 16 sarcoma, or bone sarcoma, which comprises a sub- any one of [1] to [6], wherein the giant cell tumor stance having a PPARγ-agonistic activity and/or a occurring in a bone and soft tissue is selected from PPARγ expression-inducing activity as an active in- the group consisting of giant cell tumor of bone, giant gredient. cell tumor of tendon sheath, and pigmented villon- [2] The agent for prophylactic treatment, therapeutic 5 odular synovitis. treatment, or prevention of metastasis according to [8] The agent for prophylactic treatment, therapeutic [1], wherein the substance having a PPAR γ-agonis- treatment, or prevention of metastasis according to tic activity and/or a PPARγ expression-inducing ac- any one of [1] to [7], which further contains an anti- tivity consists of one or more kinds of PPARy ago- RANKL antibody. nists selected from the group consisting of a non- 10 [9] The agent for prophylactic treatment, therapeutic steroidal anti-inflammatory agent, a thiazolidinedi- treatment, or prevention of metastasis according to one derivative, an angiotensin II receptor antagonist [8], wherein the anti-RANKL antibody is denosumab. having a PPARγ-agonistic activity, and an endog- [10] The agent for prophylactic treatment, therapeu- enous PPARγ agonist. tic treatment, or prevention of metastasis according [3] The agent for prophylactic treatment, therapeutic 15 to any one of [1] to [9], which further contains a bi- treatment, or prevention of metastasis according to sphosphonate. [1] or [2], wherein the substance having a PPARγ- [11] The agent for prophylactic treatment, therapeu- agonistic activity and/or a PPAR γ expression-induc- tic treatment, or prevention of metastasis according ing activity consists of one or more kinds of non- to [10], wherein the bisphosphonate consists of one steroidal anti-inflammatory agents selected from the 20 or more kinds of bisphosphonates selected from the group consisting of zaltoprofen, diclofenac, in- group consisting of etidronate, clodronate, tiludro- domethacin, proglumetacin, indometacin farnesil, nate, pamidronate, neridronate, olpadronate, alen- celecoxib, etodolac, meloxicam, mofezolac, dronate, ibandronate, tiludronate, incadronate, rise- acemetacin, oxaprozin, acetaminophen, lornoxi- dronate, minodronate, zoledronate, solvadronate, cam, ampiroxicam, piroxicam, naproxen, loxopro- 25 medronate, risendronate, amino-olpadronate, si- fen, rofecoxib, ethenzamide, diflunisal, aluminopro- madronate, pyridronate, rezidronate, EB1053, and fen, nabumetone, ketoprofen, acetylsalicylic acid, YH 529. ibuprofen, pranoprofen, and sulindac. [12] A local infusion for artery embolization or artifi- [4] The agent for prophylactic treatment, therapeutic cial bone, which comprises a substance having a treatment, or prevention of metastasis according to 30 PPARγ-agonistic activity and/or a PPARγ expres- any one of [1] to [3], wherein the substance having sion-inducing activity as an active ingredient. a PPARγ-agonistic activity and/or a PPARY expres- [13] The local infusion for artery embolization or ar- sion-inducing activity consists of one or more kinds tificial bone according to [12], wherein the substance of thiazolidinedione derivatives selected from the having a PPARγ-agonistic activity and/or a PPARγ group consisting of troglitazone, rosiglitazone, piogl- 35 expression-inducing activity consists of one or more itazone, balaglitazone, rivoglitazone, isaglitazone, kinds of PPARγ agonists selected from the group netoglitazone, lobeglitazone, englitazone, and cigli- consisting of a non-steroidal anti-inflammatory tazone. agent, a thiazolidinedione derivative, an angiotensin [5] The agent for prophylactic treatment, therapeutic II receptor antagonist having a PPAR γ-agonistic ac- treatment, or prevention of metastasis according to 40 tivity, and an endogenous PPARγ agonist. any one of [1] to [4], wherein the substance having [14] The local infusion for artery embolization or ar- a PPARγ-agonistic activity and/or a PPARγ expres- tificial bone according to [12] or [13], wherein the sion-inducing activity consists of one or more kinds substance having a PPAR Y-agonistic activity and/or of angiotensin II receptor antagonists having a a PPARY expression-inducing activity consists of PPARγ agonistic activity selected from the group 45 one or more kinds of non-steroidal anti-inflammatory consisting of irbesartan and telmisartan. agents selected from the group consisting of zalto- [6] The agent for prophylactic treatment, therapeutic profen, diclofenac, indomethacin, proglumetacin, in- treatment, or prevention of metastasis according to dometacin farnesil, celecoxib, etodolac, meloxicam, any one of [1] to [5], wherein the substance having mofezolac, acemetacin, oxaprozin, acetaminophen, a PPARγ-agonistic activity and/or a PPARγ expres- 50 lornoxicam, ampiroxicam, piroxicam, naproxen, sion-inducing activity consists of one or more kinds loxoprofen, rofecoxib, ethenzamide, diflunisal, alu- of endogenous PPA-RY agonists selected from the minoprofen, nabumetone, ketoprofen, acetylsalicyl- group consisting of 15-deoxy-Δ12,14-prostagladin ic acid, ibuprofen, pranoprofen, and sulindac. J2, 15-hydroxyeicosatetraenoic acid, 9-hydroxy- [15] The local infusion for artery embolization or ar- octadecadienoic acid, 13-hydroxyoctadecadienoic 55 tificial bone according to any one of [12] to [14], acid, nitrolinoleic acid, and a long chain fatty acid. wherein the substance having a PPARγ-agonistic [7] The agent for prophylactic treatment, therapeutic activity and/or a PPAR γ expression-inducing activity treatment, or prevention of metastasis according to consists of one or more kinds of thiazolidinedione

9 17 EP 3 050 573 A1 18 derivatives selected from the group consisting of tro- a combination thereof according to [22] or [23], glitazone, rosiglitazone, pioglitazone, balaglitazone, wherein the substance having a PPARγ-agonistic rivoglitazone, isaglitazone, netoglitazone, lobeglita- activity and/or a PPAR γ expression-inducing activity zone, englitazone, and ciglitazone. consists of one or more kinds of non-steroidal anti- [16] The local infusion for artery embolization or ar- 5 inflammatory agents selected from the group con- tificial bone according to any one of [12] to [15], sisting of zaltoprofen, diclofenac, indomethacin, pro- wherein the substance having a PPARγ-agonistic glumetacin, indometacin farnesil, celecoxib, etodol- activity and/or a PPAR γ expression-inducing activity ac, meloxicam, mofezolac, acemetacin, oxaprozin, consists of one or more kinds of angiotensin II re- acetaminophen, lornoxicam, ampiroxicam, piroxi- ceptor antagonists having a PPAR γ agonistic activity 10 cam, naproxen, loxoprofen, rofecoxib, ethenzamide, selected from the group consisting of irbesartan and diflunisal, aluminoprofen, nabumetone, ketoprofen, telmisartan. acetylsalicylic acid, ibuprofen, pranoprofen, and [17] The local infusion for artery embolization or ar- sulindac. tificial bone according to any one of [12] to [16], [25] The substance having a PPAR γ-agonistic activ- wherein the substance having a PPARγ-agonistic 15 ity and/or a PPARγ expression-inducing activity, or activity and/or a PPAR γ expression-inducing activity a combination thereof according to any one of [22] consists of one or more kinds of endogenous PPAR γ to [24], wherein the substance having a PPARγ-ag- agonists selected from the group consisting of 15- onistic activity and/or a PPAR γ expression-inducing deoxy-A12,14-prostagladin J2, 15-hydroxyeicosa- activity consists of one or more kinds of thiazolidin- tetraenoic acid, 9-hydroxyoctadecadienoic acid, 13- 20 edione derivatives selected from the group consist- hydroxyoctadecadienoic acid, nitrolinoleic acid, and ing of troglitazone, rosiglitazone, pioglitazone, a long chain fatty acid. balaglitazone, rivoglitazone, isaglitazone, netoglita- [18] The local infusion for artery embolization or ar- zone, lobeglitazone, englitazone, and ciglitazone. tificial bone according to any one of [12] to [17], which [26] The substance having a PPAR γ-agonistic activ- further contains an anti-RANKL antibody. 25 ity and/or a PPARγ expression-inducing activity, or [19] The local infusion for artery embolization or ar- a combination thereof according to any one of [22] tificial bone according to [18], wherein the anti-RAN- to [25], wherein the substance having a PPARγ-ag- KL antibody is denosumab. onistic activity and/or a PPAR γ expression-inducing [20] The local infusion for artery embolization or ar- activity consists of one or more kinds of angiotensin tificial bone according to any one of [12] to [19], which 30 II receptor antagonists having a PPAR γ agonistic ac- further contains a bisphosphonate. tivity selected from the group consisting of irbesartan [21] The local infusion for artery embolization or ar- and telmisartan. tificial bone according to [20], wherein the bisphos- [27] The substance having a PPAR γ-agonistic activ- phonate consists of one or more kinds of bisphos- ity and/or a PPARγ expression-inducing activity, or phonates selected from the group consisting of etid- 35 a combination thereof according to any one of [22] ronate, clodronate, tiludronate, pamidronate, nerid- to [26], wherein the substance having a PPARγ-ag- ronate, olpadronate, alendronate, ibandronate, onistic activity and/or a PPAR γ expression-inducing tiludronate, incadronate, risedronate, minodronate, activity consists of one or more kinds of endogenous zoledronate, solvadronate, medronate, risendro- PPARγ agonists selected from the group consisting nate, amino-olpadronate, simadronate, pyridronate, 40 of 15-deoxy-Δ12,14-prostagladin J2, 15-hydroxyei- rezidronate, EB1053, and YH 529. cosatetraenoic acid, 9-hydroxyoctadecadienoic ac- [22] A substance having a PPARγ-agonistic activity id, 13-hydroxyoctadecadienoic acid, nitrolinoleic ac- and/or a PPARγ expression-inducing activity, or a id, and a long chain fatty acid. combination thereof for use in prophylactic treat- [28] The substance having a PPAR γ-agonistic activ- ment, therapeutic treatment, or prevention of metas- 45 ity and/or a PPARγ expression-inducing activity, or tasis of giant cell tumor occurring in a bone and soft a combination thereof according to any one of [22] tissue, chondrosarcoma, or bone sarcoma. to [27], wherein the giant cell tumor occurring in a [23] The substance having a PPAR γ-agonistic activ- bone and soft tissue is selected from the group con- ity and/or a PPARγ expression-inducing activity, or sisting of giant cell tumor of bone, giant cell tumor of a combination thereof according to [22], which con- 50 tendonsheath, and pigmented villonodular synovitis. sists of one or more kinds of substances selected [29] The substance having a PPAR γ-agonistic activ- from the group consisting of a non-steroidal anti-in- ity and/or a PPARγ expression-inducing activity, or flammatory agent, a thiazolidinedione derivative, an a combination thereof according to any one of [22] angiotensin II receptor antagonist having a PPARγ- to [28], which further contains an anti-RANKL anti- agonistic activity, and an endogenous PPARγ ago- 55 body. nist. [30] The substance having a PPAR γ-agonistic activ- [24] The substance having a PPAR γ-agonistic activ- ity and/or a PPARγ expression-inducing activity, or ity and/or a PPARγ expression-inducing activity, or a combination thereof according to [29], wherein the

10 19 EP 3 050 573 A1 20 anti-RANKL antibody is denosumab. of thiazolidinedione derivatives selected from the [31] The substance having a PPAR Y-agonistic activ- group consisting of troglitazone, rosiglitazone, piogl- ity and/or a PPARY expression-inducing activity, or itazone, balaglitazone, rivoglitazone, isaglitazone, a combination thereof according to any one of [22] netoglitazone, lobeglitazone, englitazone, and cigli- to [30], which further contains a bisphosphonate. 5 tazone. [32] The substance having a PPAR γ-agonistic activ- [37] The method for prophylactic treatment, thera- ity and/or a PPARγ expression-inducing activity, or peutic treatment, or prevention of metastasis of giant a combination thereof according to [31], wherein the cell tumor occurring in a bone and soft tissue, chon- bisphosphonate consists of one or more kinds of bi- drosarcoma, or bone sarcoma according to any one sphosphonates selected from the group consisting 10 of [33] to [36], wherein the substance having a of etidronate, clodronate, tiludronate, pamidronate, PPARγ-agonistic activity and/or a PPARγ expres- neridronate, olpadronate, alendronate, ibandronate, sion-inducing activity consists of one or more kinds tiludronate, incadronate, risedronate, minodronate, of angiotensin II receptor antagonists having a zoledronate, solvadronate, medronate, risendro- PPARγ agonistic activity selected from the group nate, amino-olpadronate, simadronate, pyridronate, 15 consisting of irbesartan and telmisartan. rezidronate, EB1053, and YH 529. [38] The method for prophylactic treatment, thera- [33] A method for prophylactic treatment, therapeutic peutic treatment, or prevention of metastasis of giant treatment, or prevention of metastasis of giant cell cell tumor occurring in a bone and soft tissue, chon- tumor occurring in a bone and soft tissue, chondro- drosarcoma, or bone sarcoma according to any one sarcoma, or bone sarcoma, which comprises admin- 20 of [33] to [37], wherein the substance having a istering an effective amount of a substance having PPARγ-agonistic activity and/or a PPARγ expres- a PPARγ-agonistic activity and/or a PPARγ expres- sion-inducing activity consists of one or more kinds sion-inducing activity to an object. of endogenous PPARγ agonists selected from the [34] The method for prophylactic treatment, thera- group consisting of 15-deoxy-A12,14-prostagladin peutic treatment, or prevention of metastasis of giant 25 J2, 15-hydroxyeicosatetraenoic acid, 9-hydroxy- cell tumor occurring in a bone and soft tissue, chon- octadecadienoic acid, 13-hydroxyoctadecadienoic drosarcoma, or bone sarcoma according to [33], acid, nitrolinoleic acid, and a long chain fatty acid. wherein the substance having a PPARγ-agonistic [39] The method for prophylactic treatment, thera- activity and/or a PPAR γ expression-inducing activity peutic treatment, or prevention of metastasis of giant consists of one or more kinds of PPARγ agonists 30 cell tumor occurring in a bone and soft tissue, chon- selected from the group consisting of a non-steroidal drosarcoma, or bone sarcoma according to any one anti-inflammatory agent, a thiazolidinedione deriva- of [33] to [38], wherein the giant cell tumor occurring tive, an angiotensin II receptor antagonist having a in a bone and soft tissue is selected from the group PPARγ-agonistic activity, and an endogenous consisting of giant cell tumor of bone, giant cell tumor PPARγ agonist. 35 of tendon sheath, and pigmented villonodular syno- [35] The method for prophylactic treatment, thera- vitis. peutic treatment, or prevention of metastasis of giant [40] The method for prophylactic treatment, thera- cell tumor occurring in a bone and soft tissue, chon- peutic treatment, or prevention of metastasis of giant drosarcoma, or bone sarcoma according to [33] or cell tumor occurring in a bone and soft tissue, chon- [34], wherein the substance having a PPARγ-ago- 40 drosarcoma, or bone sarcoma according to any one nistic activity and/or a PPARγ expression-inducing of[33] to [39], wherein thesubstance furthercontains activity consists of one or more kinds of non-steroidal an anti-RANKL antibody. anti-inflammatory agents selected from the group [41] The method for prophylactic treatment, thera- consisting of zaltoprofen, diclofenac, indomethacin, peutic treatment, or prevention of metastasis of giant proglumetacin, indometacin farnesil, celecoxib,45 cell tumor occurring in a bone and soft tissue, chon- etodolac, meloxicam, mofezolac, acemetacin, oxa- drosarcoma, or bone sarcoma according to [40], prozin, acetaminophen, lornoxicam, ampiroxicam, wherein the anti-RANKL antibody is denosumab. piroxicam, naproxen, loxoprofen, rofecoxib, ethen- [42] The method for prophylactic treatment, thera- zamide, diflunisal, aluminoprofen, nabumetone, ke- peutic treatment, or prevention of metastasis of giant toprofen, acetylsalicylic acid, ibuprofen, pranopro- 50 cell tumor occurring in a bone and soft tissue, chon- fen, and sulindac. drosarcoma, or bone sarcoma according to any one [36] The method for prophylactic treatment, thera- of[33] to [41], wherein thesubstance furthercontains peutic treatment, or prevention of metastasis of giant a bisphosphonate. cell tumor occurring in a bone and soft tissue, chon- [43] The method for prophylactic treatment, thera- drosarcoma, or bone sarcoma according to any one 55 peutic treatment, or prevention of metastasis of giant of [33] to [35], wherein the substance having a cell tumor occurring in a bone and soft tissue, chon- PPARγ-agonistic activity and/or a PPARγ expres- drosarcoma, or bone sarcoma according to [42], sion-inducing activity consists of one or more kinds wherein the bisphosphonate consists of one or more

11 21 EP 3 050 573 A1 22 kinds of bisphosphonates selected from the group an arteriosclerosis-related gene, and an an- consisting of etidronate, clodronate, tiludronate, pa- ti-inflammation-related gene, midronate, neridronate, olpadronate, alendronate, (g) amount of lipid contained in a fat cell or ibandronate, tiludronate, incadronate, risedronate, fat tissue, minodronate, zoledronate, solvadronate, medro- 5 nate, risendronate, amino-olpadronate, simadro- and nate, pyridronate, rezidronate, EB1053, and YH 529. (3) the step of selecting a test substance that [44] A method for screening for an agent for prophy- changes a value or values of the intracellular lactic treatment, therapeutic treatment, or prevention index or indices in the presence of a test sub- of metastasis of giant cell tumor occurring in a bone 10 stance compared with the value or values of the and soft tissue, chondrosarcoma, or bone sarcoma, intracellular index or indices observed in the ab- which comprises the following steps: sence of the test substance.

(1) the step of culturing a cell or tissue derived [45] The screening method according to [44], where- from giant cell tumor occurring in a bone and 15 in the giant cell tumor occurring in a bone and soft soft tissue, chondrosarcoma, or bone sarcoma tissue is selected from the group consisting of giant in the presence or absence of a test substance, cell tumor of bone, giant cell tumor of tendon sheath, (2) the step of measuring one or more kinds of and pigmented villonodular synovitis. indices selected from the group consisting of those defined in (a) to (g) mentioned below in 20 Effect of the Invention the presence or absence of the test substance; [0066] With the agent for prophylactic treatment, ther- (a) one or more indices selected from the apeutic treatment, or prevention of metastasis of the group consisting of gene expression present invention, giant cell tumor occurring in a bone amount of PPARγ, and protein amount 25 and soft tissue, chondrosarcoma, or bone sarcoma can (b) one or more indices selected from the be treated without carrying out surgical operation. Where group consisting of gene expression the agent for prophylactic treatment, therapeutic treat- amount of an apoptosis-related gene, pro- ment, or prevention of metastasis of the present invention tein amount of a translation product of an is used with ablative operation of giant cell tumor occur- apoptosis-related gene, and biological ac- 30 ring in a bone and soft tissue, chondrosarcoma, or bone tivity of a translation product of an apopto- sarcoma, recurrence or metastasis of the tumor caused sis-related gene, by tumor that could not be excised can be prevented. (c) one or more indices selected from the Furthermore, the agent for prophylactic treatment, ther- group consisting of gene expression apeutic treatment, or prevention of metastasis of the amount of a fat cell differentiation-related 35 present invention can be orally administered. gene, protein amount of a translation prod- [0067] The agent for prophylactic treatment, therapeu- uct of a fat cell differentiation-related gene, tic treatment, or prevention of metastasis and the local and biological activity of a translation prod- infusion for artery embolization or the artificial bone of uct of a fat cell differentiation-related gene, the present invention are a radical therapeutic agent or (d) one or more indices selected from the 40 radical therapeutic material that can cause apoptosis in group consisting of gene expression giant cell tumor occurring in a bone and soft tissue, chon- amount of an arteriosclerosis-related gene, drosarcoma, or bone sarcoma to make the tumor disap- protein amount of a translation product of pear, and can induce differentiation of the tumor into fat an arteriosclerosis-related gene, and bio- cells to make the tumor disappear. The agent for prophy- logical activity of a translation product of an 45 lactic treatment, therapeutic treatment, or prevention of arteriosclerosis-related gene, metastasis, and the local infusion for artery embolization, (e) one or more indices selected from the orthe artificialbone of thepresent inventioncan suppress group consisting of gene expression recurrence and metastasis of giant cell tumor occurring amount of an anti-inflammation-related in a bone and soft tissue, chondrosarcoma, or bone sar- gene, protein amount of a translation prod- 50 coma. The agent for prophylactic treatment, therapeutic uct of an anti-inflammation-related gene, treatment, or prevention of metastasis, and the local in- and biological activity of a translation prod- fusion for artery embolization, or the artificial bone of the uct of an anti-inflammation-related gene, present invention can restore motor functions of patients (f) an index consisting of a PPAR γ-agonistic of giant cell tumor occurring in a bone and soft tissue, activity that can promote transcription of55 chondrosarcoma, or bone sarcoma, prevent nerve dam- one or more kinds of genes selected from ages in the patients, and can dramatically improve quality the group consisting of an apoptosis-related of life of the patients. gene, a fat cell differentiation-related gene, [0068] Even if tumor cannot be disappeared with the

12 23 EP 3 050 573 A1 24 agent for prophylactic treatment, therapeutic treatment, [Fig. 3] A graph showing ratios of Tunel-positive cells or prevention of metastasis, the local infusion for artery in GCTB cultured cells that were cultured in a zalto- embolization, or the artificial bone of the present inven- profen-containing medium. The concentrations are tion can improve or maintain the ability of patients to carry the concentrations of zaltoprofen, and the times are out everyday activities. Even if tumor cannot be disap- 5 the times of culture performed in the presence of peared with the agent for prophylactic treatment, thera- zaltoprofen. The vertical axis represents the ratio of peutic treatment, or prevention of metastasis, the local Tunel-positive cells. The numerals on the right side infusion for artery embolization, or the artificial bone of of P< indicate the significance levels obtained as a the present invention can promote restoration, formation, result of statistical analysis. or hardening of bones to improve or maintain motor func- 10 [Fig. 4] Photographs showing results of caspase 3 tions. staining of GCTB cultured cells that were cultured in [0069] When the therapeutic agent or therapeutic ma- a zaltoprofen-containing medium. The concentra- terial of the present invention contains a non-steroidal tions are the concentrations of zaltoprofen, and the anti-inflammatory agent, it also has efficacies for amel- times are the times of culture performed in the pres- iorating inflammation of affected part and reducing pain 15 ence of zaltoprofen. The indication of DAPI means of the affected part. that the results are results of nuclear staining with [0070] The agent for prophylactic treatment, therapeu- DAPI, which is a fluorescent dye. tic treatment, or prevention of metastasis of the present [Fig. 5] A graph showing ratios of caspase 3-positive invention can also be used as a chemotherapeutic agent cells in GCTB cultured cells that were cultured in a to be used before or after a surgical operation. 20 zaltoprofen-containing medium. The concentrations [0071] Moreover, according to the present invention, are the concentrations of zaltoprofen, and the times by selecting a test substance that controls PPARY and are the times of culture performed in the presence apoptosis or differentiation into fat cells, a novel agent of zaltoprofen. The vertical axis represents the ratio for prophylactic treatment, therapeutic treatment, or pre- ofcaspase 3-positive cells.The numerals on theright vention of metastasis of giant cell tumor occurring in a 25 side of P< indicate the significance levels obtained bone and soft tissue, chondrosarcoma, or bone sarcoma as a result of statistical analysis. can be searched for. [Fig. 6]Photographs showingresults of PPARy stain- ing of GCTB cultured cells that were cultured in a Brief Description of the Drawings zaltoprofen-containing medium. The concentrations 30 are the concentrations of zaltoprofen. The indication [0072] of DAPI means that the results are results of nuclear staining with DAPI, which is a fluorescent dye. [Fig. 1] Graphs showing results of suppression of [Fig. 7] A graph showing ratios of PPARγ-positive proliferation of GCTB cultured cells that were cul- cells in GCTB cultured cells that were cultured in a tured in a zaltoprofen-containing medium. The indi- 35 zaltoprofen-containing medium. The concentrations cations of GCT case 1 and GCT case 2 mean giant indicated under the horizontal axis are the concen- cell tumor of bone cultured cells from different ori- trations of zaltoprofen. The vertical axis represents gins. The indication of WST assay means that the the ratio of PPARγ-positive cells. The numerals on cell proliferation was measured by using Cell Count- the right side of P< indicate the significance levels ing Kit-8 (CCK-8, Dojindo) using a tetrazolium salt 40 obtained as a result of statistical analysis. WST-8 as a chromophoric substrate. The horizontal [Fig. 8] Photographs showing results of lipid staining axes represent the concentration of zaltoprofen, and of GCTB cultured cells that were cultured in a zalto- the vertical axes represent the live cell count meas- profen-containing medium. The concentrations are ured on the basis of absorbance at 450 nm. The nu- the concentrations of zaltoprofen. The indication of merals on the right side of P< indicate the signifi- 45 DAPI means that the results are results of nuclear cance levels obtained as a result of statistical anal- staining with DAPI, which is a fluorescent dye. ysis. [Fig. 9] A graph showing ratios of lipid-positive cells [Fig. 2] Photographs showing results of Tunel assay in GCTB cultured cells that were cultured in a zalto- performed with GCTB cultured cells that were cul- profen-containing medium. The concentrations indi- tured in a zaltoprofen-containing medium. In the50 cated under the horizontal axis are the concentra- Tunel assay, fragmented DNAs produced in the tions of zaltoprofen. The vertical axis represents the process of apoptosis were detected by the TdT-me- ratio of lipid-positive cells. The concentrations are diated dUTP nick end labeling method (TUNEL). The the concentrations of zaltoprofen. The numerals on concentrations are the concentrations of zaltopro- the right side of P< indicate the significance levels fen, and the times are the times of culture performed 55 obtained as a result of statistical analysis. in the presence of zaltoprofen. The indication of [Fig. 10]Photographs showing results ofTunel assay DAPI means that the results are results of nuclear and caspase 3 staining of GCTB samples derived staining with DAPI, which is a fluorescent dye. from a GCTB patient administered with zaltoprofen.

13 25 EP 3 050 573 A1 26

The indication of DAPI means that the results are of WST assay means that the cell proliferation was results of nuclear staining with DAPI, which is a flu- measured by using Cell Counting Kit-8 (CCK-8, orescent dye. Dojindo) using a tetrazolium salt WST-8 as a [Fig. 11] Photographs showing results of PPARy chromophoric substrate. The concentrations indicat- staining of GCTB samples derived from a GCTB pa- 5 ed under the horizontal axes are the concentrations tient administered with zaltoprofen. The indication of of the drugs, and the vertical axes represent the live DAPI means that the results are results of nuclear cell count measured on the basis of absorbance at staining with DAPI, which is a fluorescent dye. 450 nm. The numerals on the right side of P indicate [Fig. 12] Graphs showing results of suppression of the significance levels obtained as a result of statis- proliferation of GCTB cultured cells that were cul- 10 tical analysis. tured in an acetaminophen, indomethacin, di- [Fig. 16] Graphs showing results of suppression of clofenac, or troglitazone-containing medium. The in- proliferation of cultured cells derived from giant cell dication of GCT case 1 represents the origin of the tumor of tendon sheath (GCTT) or cultured cells de- cultured cells of giant cell tumor of bone. The indi- rived from pigmented villonodular synovitis (PVNS) cation of WST assay means that the cell proliferation 15 cultured in a zaltoprofen-containing medium. The in- was measured by using Cell Counting Kit-8 (CCK- dication of WST assay means that the cell prolifer- 8, Dojindo) using a tetrazolium salt WST-8 as a ation was measured by using Cell Counting Kit-8 chromophoric substrate. The horizontal axes repre- (CCK-8, Dojindo) using a tetrazolium salt WST-8 as sent the concentration of each drug, and the vertical a chromophoric substrate. The horizontal axes rep- axes represent the live cell count measured on the 20 resent the concentration of zaltoprofen, and the ver- basis of absorbance at 450 nm. The numerals on tical axes represent the live cell count measured on the right side of P< indicate the significance levels the basis of absorbance at 450 nm. The numerals obtained as a result of statistical analysis. on the right side of P< indicate the significance levels [Fig. 13] Graphs showing results of suppression of obtained as a result of statistical analysis. proliferation of GCTB cultured cells that were cul- 25 [Fig. 17] Graphs showing results of suppression of tured in an acetaminophen, indomethacin, di- proliferation of cultured cells derived from giant cell clofenac, or troglitazone-containing medium. The in- tumor of tendon sheath (GCTT) or cultured cells de- dication of GCT case 2 represents the origin of the rived from pigmented villonodular synovitis (PVNS) cultured cells of giant cell tumor of bone. The indi- cultured in a troglitazone-containing medium. The cation of WST assay means that the cell proliferation 30 indication of WST assay means that the cell prolif- was measured by using Cell Counting Kit-8 (CCK- eration was measured by using Cell Counting Kit-8 8, Dojindo) using a tetrazolium salt WST-8 as a (CCK-8, Dojindo) using a tetrazolium salt WST-8 as chromophoric substrate. The horizontal axes repre- a chromophoric substrate. The horizontal axes rep- sent the concentration of each drug, and the vertical resent the concentration of troglitazone, and the ver- axes represent the live cell count measured on the 35 tical axes represent the live cell count measured on basis of absorbance at 450 nm. The numerals on the basis of absorbance at 450 nm. The numerals the right side of P< indicate the significance levels on the right side of P< indicate the significance levels obtained as a result of statistical analysis. obtained as a result of statistical analysis. [Fig. 14] Graphs showing effect of PPARy siRNA on [Fig. 18]Photographs showing results ofTunel assay GCTB cultured cells that were cultured in a zaltopro- 40 of cultured cells derived from giant cell tumor of ten- fen or troglitazone-containing medium. The indica- don sheath (GCTT) cultured in a zaltoprofen or tro- tion of GCT case 1 represents the origin of the cul- glitazone-containing medium. In the Tunel assay, tured cells of giant cell tumor of bone. The indication fragmented DNAs produced in the process of apop- of WST assay means that the cell proliferation was tosis were detected by the TdT-mediated dUTP nick measured by using Cell Counting Kit-8 (CCK-8,45 end labeling method (TUNEL). The indication of Dojindo) using a tetrazolium salt WST-8 as a DAPI means that the results are results of nuclear chromophoric substrate. The concentrations indicat- staining with DAPI, which is a fluorescent dye. ed under the horizontal axes are the concentrations [Fig. 19] Graphs showing ratios of Tunel-positive of the drugs, and the vertical axes represent the live cells in cultured cells derived from giant cell tumor cell count measured on the basis of absorbance at 50 of tendon sheath (GCTT) cultured in a zaltoprofen 450 nm. The numerals on the right side of P indicate or troglitazone-containing medium. The vertical axes the significance levels obtained as a result of statis- represent the ratio of Tunel-positive cells, and the tical analysis. concentrations indicated under the horizontal axes [Fig. 15] Graphs showing effect of PPARy siRNA on are the concentrations of the drugs. The numerals GCTB cultured cells that were cultured in a zaltopro- 55 on the right side of P indicate the significance levels fen or troglitazone-containing medium. The indica- obtained as a result of statistical analysis. tion of GCT case 2 represents the origin of the cul- [Fig. 20]Photographs showing results ofTunel assay tured cells of giant cell tumor of bone. The indication of cultured cells derived from pigmented villonodular

14 27 EP 3 050 573 A1 28 synovitis (PVNS) cultured in a zaltoprofen or trogli- are the concentrations of the drugs. The numerals tazone-containing medium. In the Tunel assay, frag- on the right side of P indicate the significance levels mented DNAs produced in the process of apoptosis obtained as a result of statistical analysis. were detected by the TdT-mediated dUTP nick end [Fig. 28] Photographs showing results of PPARy labeling method (TUNEL). The indication of DAPI 5 staining of cultured cells derived from pigmented vil- means that the results are results of nuclear staining lonodular synovitis (PVNS) cultured in a zaltoprofen with DAPI, which is a fluorescent dye. or troglitazone-containing medium. The indication of [Fig. 21] Graphs showing ratios of Tunel-positive DAPI means that the results are results of nuclear cells in cultured cells derived from pigmented villon- staining with DAPI, which is a fluorescent dye. odular synovitis (PVNS) cultured in a zaltoprofen or 10 [Fig. 29] Graphs showing ratios of PPARγ-positive troglitazone-containing medium. The vertical axes cells in cultured cells derived from pigmented villon- represent the ratio of Tunel-positive cells, and the odular synovitis (PVNS) cultured in a zaltoprofen or concentrations indicated under the horizontal axes troglitazone-containing medium. The vertical axes are the concentrations of the drugs. The numerals represent the ratio of PPARγ-positive cells, and the on the right side of P indicate the significance levels 15 concentrations indicated under the horizontal axes obtained as a result of statistical analysis. are the concentrations of the drugs. The numerals [Fig. 22] Photographs showing results of caspase 3 on the right side of P indicate the significance levels staining of cultured cells derived from giant cell tumor obtained as a result of statistical analysis. of tendon sheath (GCTT) cultured in a zaltoprofen [Fig. 30] Photographs showing results of lipid stain- or troglitazone-containing medium. The indication of 20 ing of cultured cells derived from giant cell tumor of DAPI means that the results are results of nuclear tendon sheath (GCTT) or cultured cells derived from staining with DAPI, which is a fluorescent dye. pigmented villonodular synovitis (PVNS) cultured in [Fig.23] Graphs showing ratios of caspase 3-positive a zaltoprofen-containing medium. The indication of cells in cultured cells derived from giant cell tumor DAPI means that the results are results of nuclear of tendon sheath (GCTT) cultured in a zaltoprofen 25 staining with DAPI, which is a fluorescent dye. or troglitazone-containing medium. The vertical axes [Fig. 31] Graphs showing ratios of lipid-positive cells represent the ratio of caspase 3-positive cells, and in cultured cells derived from giant cell tumor of ten- the concentrations indicated under the horizontal ax- don sheath (GCTT) or cultured cells derived from es are the concentrations of the drugs. The numerals pigmented villonodular synovitis (PVNS) cultured in on the right side of P indicate the significance levels 30 a zaltoprofen-containing medium. The vertical axes obtained as a result of statistical analysis. represents the ratio of lipid-positive cells, and the [Fig. 24] Photographs showing results of caspase 3 concentrations indicated under the horizontal axes staining of cultured cells derived from pigmented vil- are the concentrations of the drugs. The numerals lonodular synovitis (PVNS) cultured in a zaltoprofen on the right side of P indicate the significance levels or troglitazone-containing medium. The indication of 35 obtained as a result of statistical analysis. DAPI means that the results are results of nuclear [Fig. 32] Graphs showing results of suppression of staining with DAPI, which is a fluorescent dye. proliferation of cultured cells derived from giant cell [Fig.25] Graphs showing ratios of caspase 3-positive tumor of bone (GCTB), cultured cells derived from cells in cultured cells derived from pigmented villon- giant cell tumor of tendon sheath (GCTT), and cul- odular synovitis (PVNS) cultured in a zaltoprofen or 40 tured cells derived from pigmented villonodular syn- troglitazone-containing medium. The vertical axes ovitis (PVNS) cultured in a pioglitazone-containing represent the ratio of caspase 3-positive cells, and medium. The indications of CT case 1 and GCT case the concentrations indicated under the horizontal ax- 2 represent cultured cells of giant cell tumor of bone es are the concentrations of the drugs. The numerals from different origins. The horizontal axes represent on the right side of P indicate the significance levels 45 the concentration of pioglitazone, and the vertical obtained as a result of statistical analysis. axes represent the live cell count measured on the [Fig. 26] Photographs showing results of PPARy basis of absorbance at 450 nm using Cell Counting staining of cultured cells derived from giant cell tumor Kit-8 (CCK-8, Dojindo) using a tetrazolium salt WST- of tendon sheath (GCTT) cultured in a zaltoprofen 8 as a chromophoric substrate. The numerals on the or troglitazone-containing medium. The indication of 50 right side of P< indicate the significance levels ob- DAPI means that the results are results of nuclear tained as a result of statistical analysis. staining with DAPI, which is a fluorescent dye. [Fig. 33] A photograph showing an X-ray image of [Fig. 27] Graphs showing ratios of PPARγ-positive the pelvic part of a patient with recurrence of giant cells in cultured cells derived from giant cell tumor cell tumor of bone (GCTB) in the pelvic part obtained of tendon sheath (GCTT) cultured in a zaltoprofen 55 before the administration of zaltoprofen. This case or troglitazone-containing medium. The vertical axes is the same as the case a mentioned in the table of represent the ratio of PPARγ-positive cells, and the Fig. 79. concentrations indicated under the horizontal axes [Fig. 34] A photograph showing an MRI image of the

15 29 EP 3 050 573 A1 30 pelvic part of a patient with recurrence of giant cell of the right knee part of a patient with recurrence of tumor of bone (GCTB) in the pelvic part obtained pigmented villonodular synovitis (PVNS) in the right before the administration of zaltoprofen. This case knee part obtained after two months of the adminis- is the same as the case a mentioned in the table of tration of zaltoprofen. The arrows indicate shrinkage Fig. 79. 5 of tumor. This case is the same as the case b men- [Fig. 35] A photograph showing an MRI image of the tioned in the table of Fig. 86. pelvic part of a patient with recurrence of giant cell [Fig. 44] A photograph showing a transversal MRI tumor of bone (GCTB) in the pelvic part obtained image of the right knee part of a patient with recur- after two months of the administration of zaltoprofen. rence of pigmented villonodular synovitis (PVNS) in The arrows indicate a tumor necrosis region. This 10 the right knee part obtained after two months of the case is the same as the case a mentioned in the administration of zaltoprofen. The arrows indicate table of Fig. 79. shrinkage of tumor. This case is the same as the [Fig. 36] A photograph showing an MRI image of the case b mentioned in the table of Fig. 86. pelvic part of a patient with recurrence of giant cell [Fig. 45] Graphs showing results of suppression of tumor of bone (GCTB) in the pelvic part obtained 15 proliferation of cells of a chondrosarcoma-derived after four months of the administration of zaltoprofen. cell line (H-EMC-SS) cultured in a zaltoprofen, tro- The arrows indicate a tumor necrosis region. This glitazone, or pioglitazone-containing medium. The case is the same as the case a mentioned in the cell proliferation was measured by using Cell Count- table of Fig. 79. ing Kit-8 (CCK-8, Dojindo) using a tetrazolium salt [Fig. 37] A photograph showing a sagittal MRI image 20 WST-8 as a chromophoric substrate. The concen- of the right knee part of a patient with recurrence of trations indicated under the horizontal axes are the pigmented villonodular synovitis (PVNS) in the right concentrations of the drugs, and the vertical axis rep- knee part obtained before the administration of zal- resent the live cell count measured on the basis of toprofen. This case is the same as the case i men- absorbance at 450 nm. The numerals on the right tioned in the table of Fig. 86. 25 side of P< indicate the significance levels obtained [Fig. 38] A photograph showing a transversal MRI as a result of statistical analysis. image of the right knee part of a patient with recur- [Fig. 46] Photographs showing results of caspase 3 rence of pigmented villonodular synovitis (PVNS) in staining of cells of a chondrosarcoma-derived cell the right knee part obtained before the administration line (H-EMC-SS) cultured in a zaltoprofen, troglita- of zaltoprofen. This case is the same as the case i 30 zone, or pioglitazone-containing medium. The indi- mentioned in the table of Fig. 86. cation of DAPI means that the results are results of [Fig. 39] A photograph showing a sagittal MRI image nuclear staining with DAPI, which is a fluorescent of the right knee part of a patient with recurrence of dye. pigmented villonodular synovitis (PVNS) in the right [Fig. 47]Graphs showing ratiosof caspase 3-positive knee part obtained after three months of the admin- 35 cells in cells of a chondrosarcoma-derived cell line istration of zaltoprofen. The arrows indicate attenu- (H-EMC-SS) cultured in a zaltoprofen, troglitazone, ation of the MRI imaging effect. This case is the same or pioglitazone-containing medium. The vertical ax- as the case i mentioned in the table of Fig. 86. es represent the ratio of caspase 3-positive cells, [Fig. 40] A photograph showing a transversal MRI and the concentrations indicated under the horizon- image of the right knee part of a patient with recur- 40 tal axes are the concentrations of the drugs. The rence of pigmented villonodular synovitis (PVNS) in numerals on the right side of P indicate the signifi- the right knee part obtained after three months of the cance levels obtained as a result of statistical anal- administration of zaltoprofen. The arrows indicate at- ysis. tenuation of the MRI imaging effect. This case is the [Fig. 48] Photographs showing results of PPARy same as the case i mentioned in the table of Fig. 86. 45 staining of cells of a chondrosarcoma-derived cell [Fig. 41] A photograph showing a coronal MRI image line (H-EMC-SS) cultured in a zaltoprofen, troglita- of the right knee part of a patient with recurrence of zone, or pioglitazone-containing medium. The indi- pigmented villonodular synovitis (PVNS) in the right cation of DAPI means that the results are results of knee part obtained before the administration of zal- nuclear staining with DAPI, which is a fluorescent toprofen. This case is the same as the case b men- 50 dye. tioned in the table of Fig. 86. [Fig. 49] Graphs showing ratios of PPARγ-positive [Fig. 42] A photograph showing a transversal MRI cells in cells of a chondrosarcoma-derived cell line image of the right knee part of a patient with recur- (H-EMC-SS) cultured in a zaltoprofen, troglitazone, rence of pigmented villonodular synovitis (PVNS) in or pioglitazone-containing medium. The vertical ax- the right knee part obtained before the administration 55 es represent the ratio of PPARγ-positive cells, and of zaltoprofen. This case is the same as the case b the concentrations indicated under the horizontal ax- mentioned in the table of Fig. 86. es are the concentrations of the drugs. The numerals [Fig. 43] A photograph showing a coronal MRI image on the right side of P indicate the significance levels

16 31 EP 3 050 573 A1 32 obtained as a result of statistical analysis. sarcoma cell lines (H-EMC-SS, SW1353), which [Fig. 50] Graphs showing results of suppression of were cultured in a diclofenac-containing medium. proliferation of cultured cells of giant cell tumor of The horizontal axes represent the concentration of bone (GCT-1, GCT-2), cultured cells of giant cell tu- the drug, and the vertical axes represent the live cell mor of tendon sheath (GCTT), cultured cells of pig- 5 count measured on the basis of absorbance at 450 mented villonodular synovitis, and cells of chondro- nm using CCK-8 (Dojindo). The numerals on the right sarcoma cell lines (H-EMC-SS, SW1353), which side of P< indicate the significance levels obtained were cultured in a pioglitazone-containing medium. as a result of statistical analysis. The horizontal axes represent the concentration of [Fig. 55] Graphs showing results of suppression of the drug, and the vertical axes represent the live cell 10 proliferation of cultured cells of giant cell tumor of count measured on the basis of absorbance at 450 bone (GCT-1, GCT-2), cultured cells of giant cell tu- nmusing CCK-8 (Dojindo). The numeralson the right mor of tendon sheath (GCTT), cultured cells of pig- side of P< indicate the significance levels obtained mented villonodular synovitis, and cells of chondro- as a result of statistical analysis. sarcoma cell lines (H-EMC-SS, SW1353), which [Fig. 51] Graphs showing results of suppression of 15 were cultured in an indomethacin-containing medi- proliferation of cultured cells of giant cell tumor of um. The horizontal axes represent the concentration bone (GCT-1, GCT-2), cultured cells of giant cell tu- of the drug, and the vertical axes represent the live mor of tendon sheath (GCTT), cultured cells of pig- cell count measured on the basis of absorbance at mented villonodular synovitis, and cells of chondro- 450 nm using CCK-8 (Dojindo). The numerals on the sarcoma cell lines (H-EMC-SS, SW1353), which20 right side of P< indicate the significance levels ob- were cultured in a troglitazone-containing medium. tained as a result of statistical analysis. The horizontal axes represent the concentration of [Fig. 56] Graphs showing results of suppression of the drug, and the vertical axes represent the live cell proliferation of cultured cells of giant cell tumor of count measured on the basis of absorbance at 450 bone (GCT-1, GCT-2), cultured cells of giant cell tu- nmusing CCK-8 (Dojindo). The numeralson the right 25 mor of tendon sheath (GCTT), cultured cells of pig- side of P< indicate the significance levels obtained mented villonodular synovitis, and cells of chondro- as a result of statistical analysis. sarcoma cell lines (H-EMC-SS, SW1353), which [Fig. 52] Graphs showing results of suppression of werecultured ina celecoxib-containingmedium. The proliferation of cultured cells of giant cell tumor of horizontal axes represent the concentration of the bone (GCT-1, GCT-2), cultured cells of giant cell tu- 30 drug, and the vertical axes represent the live cell mor of tendon sheath (GCTT), cultured cells of pig- count measured on the basis of absorbance at 450 mented villonodular synovitis, and cells of chondro- nm using CCK-8 (Dojindo). The numerals on the right sarcoma cell lines (H-EMC-SS, SW1353), which side of P< indicate the significance levels obtained were cultured in a rosiglitazone-containing medium. as a result of statistical analysis. The horizontal axes represent the concentration of 35 [Fig. 57] Graphs showing results of suppression of the drug, and the vertical axes represent the live cell proliferation of cultured cells of giant cell tumor of count measured on the basis of absorbance at 450 bone (GCT-1, GCT-2), cultured cells of giant cell tu- nmusing CCK-8 (Dojindo). The numeralson the right mor of tendon sheath (GCTT), cultured cells of pig- side of P< indicate the significance levels obtained mented villonodular synovitis, and cells of chondro- as a result of statistical analysis. 40 sarcoma cell lines (H-EMC-SS, SW1353), which [Fig. 53] Graphs showing results of suppression of were cultured in an etodolac-containing medium. proliferation of cultured cells of giant cell tumor of The horizontal axes represent the concentration of bone (GCT-1, GCT-2), cultured cells of giant cell tu- the drug, and the vertical axes represent the live cell mor of tendon sheath (GCTT), cultured cells of pig- count measured on the basis of absorbance at 450 mented villonodular synovitis, and cells of chondro- 45 nm using CCK-8 (Dojindo). The numerals on the right sarcoma cell lines (H-EMC-SS, SW1353), which side of P< indicate the significance levels obtained were cultured in a zaltoprofen-containing medium. as a result of statistical analysis. The horizontal axes represent the concentration of [Fig. 58] Graphs showing results of suppression of the drug, and the vertical axes represent the live cell proliferation of cultured cells of giant cell tumor of count measured on the basis of absorbance at 450 50 bone (GCT-1, GCT-2), cultured cells of giant cell tu- nmusing CCK-8 (Dojindo). The numeralson the right mor of tendon sheath (GCTT), cultured cells of pig- side of P< indicate the significance levels obtained mented villonodular synovitis, and cells of chondro- as a result of statistical analysis. sarcoma cell lines (H-EMC-SS, SW1353), which [Fig. 54] Graphs showing results of suppression of were cultured in a meloxicam-containing medium. proliferation of cultured cells of giant cell tumor of 55 The horizontal axes represent the concentration of bone (GCT-1, GCT-2), cultured cells of giant cell tu- the drug, and the vertical axes represent the live cell mor of tendon sheath (GCTT), cultured cells of pig- count measured on the basis of absorbance at 450 mented villonodular synovitis, and cells of chondro- nm using CCK-8 (Dojindo). The numerals on the right

17 33 EP 3 050 573 A1 34 side of P< indicate the significance levels obtained mented villonodular synovitis, and cells of chondro- as a result of statistical analysis. sarcoma cell lines (H-EMC-SS, SW1353), which [Fig. 59] Graphs showing results of suppression of were cultured in a lornoxicam-containing medium. proliferation of cultured cells of giant cell tumor of The horizontal axes represent the concentration of bone (GCT-1, GCT-2), cultured cells of giant cell tu- 5 the drug, and the vertical axes represent the live cell mor of tendon sheath (GCTT), cultured cells of pig- count measured on the basis of absorbance at 450 mented villonodular synovitis, and cells of chondro- nm using CCK-8 (Dojindo). The numerals on the right sarcoma cell lines (H-EMC-SS, SW1353), which side of P< indicate the significance levels obtained were cultured in a mofezolac-containing medium. as a result of statistical analysis. The horizontal axes represent the concentration of 10 [Fig. 64] Graphs showing results of suppression of the drug, and the vertical axes represent the live cell proliferation of cultured cells of giant cell tumor of count measured on the basis of absorbance at 450 bone (GCT-1, GCT-2), cultured cells of giant cell tu- nmusing CCK-8 (Dojindo). The numeralson the right mor of tendon sheath (GCTT), cultured cells of pig- side of P< indicate the significance levels obtained mented villonodular synovitis, and cells of chondro- as a result of statistical analysis. 15 sarcoma cell lines (H-EMC-SS, SW1353), which [Fig. 60] Graphs showing results of suppression of were cultured in an ampiroxicam-containing medi- proliferation of cultured cells of giant cell tumor of um. The horizontal axes represent the concentration bone (GCT-1, GCT-2), cultured cells of giant cell tu- of the drug, and the vertical axes represent the live mor of tendon sheath (GCTT), cultured cells of pig- cell count measured on the basis of absorbance at mented villonodular synovitis, and cells of chondro- 20 450 nm using CCK-8 (Dojindo). The numerals on the sarcoma cell lines (H-EMC-SS, SW1353), which right side of P< indicate the significance levels ob- were cultured in an acemetacin-containing medium. tained as a result of statistical analysis. The horizontal axes represent the concentration of [Fig. 65] Graphs showing results of suppression of the drug, and the vertical axes represent the live cell proliferation of cultured cells of giant cell tumor of count measured on the basis of absorbance at 450 25 bone (GCT-1, GCT-2), cultured cells of giant cell tu- nmusing CCK-8 (Dojindo). The numeralson the right mor of tendon sheath (GCTT), cultured cells of pig- side of P< indicate the significance levels obtained mented villonodular synovitis, and cells of chondro- as a result of statistical analysis. sarcoma cell lines (H-EMC-SS, SW1353), which [Fig. 61] Graphs showing results of suppression of werecultured ina naproxen-containingmedium. The proliferation of cultured cells of giant cell tumor of 30 horizontal axes represent the concentration of the bone (GCT-1, GCT-2), cultured cells of giant cell tu- drug, and the vertical axes represent the live cell mor of tendon sheath (GCTT), cultured cells of pig- count measured on the basis of absorbance at 450 mented villonodular synovitis, and cells of chondro- nm using CCK-8 (Dojindo). The numerals on the right sarcoma cell lines (H-EMC-SS, SW1353), which side of P< indicate the significance levels obtained were cultured in an oxaprozin-containing medium. 35 as a result of statistical analysis. The horizontal axes represent the concentration of [Fig. 66] Graphs showing results of suppression of the drug, and the vertical axes represent the live cell proliferation of cultured cells of chondrosarcoma count measured on the basis of absorbance at 450 (OUMS-27) cultured in a zaltoprofen, rosiglitazone, nmusing CCK-8 (Dojindo). The numeralson the right or troglitazone-containing medium. The concentra- side of P< indicate the significance levels obtained 40 tions indicated under the horizontal axes are the con- as a result of statistical analysis. centrations of the drugs, and the vertical axes rep- [Fig. 62] Graphs showing results of suppression of resent the live cell count measured on the basis of proliferation of cultured cells of giant cell tumor of absorbance at 450 nm using CCK-8 (Dojindo). bone (GCT-1, GCT-2), cultured cells of giant cell tu- [Fig. 67] Graphs showing results of suppression of mor of tendon sheath (GCTT), cultured cells of pig- 45 proliferation of cultured cells of chondrosarcoma mented villonodular synovitis, and cells of chondro- (OUMS-27) cultured in a zaltoprofen, rosiglitazone, sarcoma cell lines (H-EMC-SS, SW1353), which or troglitazone-containing medium. The concentra- were cultured in an acetaminophen-containing me- tions indicated under the horizontal axes are the con- dium. The horizontal axes represent the concentra- centrations of the drugs, and the vertical axes rep- tion of the drug, and the vertical axes represent the 50 resent the live cell count measured on the basis of live cell count measured on the basis of absorbance absorbance at 450 nm using CCK-8 (Dojindo). The at 450 nm using CCK-8 (Dojindo). The numerals on black bars represent the live cell counts obtained the right side of P< indicate the significance levels after the cells were incubated with 1 mM (final con- obtained as a result of statistical analysis. centration) of GW9662 (Sigma-Aldrich, M6191), [Fig. 63] Graphs showing results of suppression of 55 which is an irreversible antagonist and then zalto- proliferation of cultured cells of giant cell tumor of profen, rosiglitazone, or troglitazone was added. The bone (GCT-1, GCT-2), cultured cells of giant cell tu- white bars represent the live cell counts obtained mor of tendon sheath (GCTT), cultured cells of pig- after the cells were incubated only with DMSO not

18 35 EP 3 050 573 A1 36 containing GW9662, and then zaltoprofen, rosiglita- taining medium. The experimental groups are shown zone, or troglitazone was added. under the horizontal axis, and the vertical axis rep- [Fig. 68] A graph showing results of suppression of resent the live cell count measured on the basis of proliferation of cultured cells of chondrosarcoma absorbance at 450 nm using CCK-8 (Dojindo). From (SW1353), which were cultured in a zaltoprofen-con- 5 the left, the group indicated as "Si Control" is a group taining medium. Settings of the drugs are indicated for which PPARγ gene expression was suppressed under the horizontal axis, and the vertical axis rep- with PPARγ-siRNA, and then the cells were cultured resent the live cell count measured on the basis of with DMSO as a vehicle control, the group indicated absorbance at 450 nm using CCK-8 (Dojindo). The as "Si Zalto" is a group for which PPARγ gene ex- black bars represent the live cell counts obtained 10 pression was suppressed with PPARγ-siRNA, and after the cells were incubated with 1 mM (final con- then the cells were cultured with zaltoprofen, the centration) of GW9662, which is an irreversible an- group indicated as "SiN Control" is a control group tagonist and then zaltoprofen was added. The white for which the cells were infected with negative-siR- bars represent the live cell counts obtained after the NA, and then cultured with DMSO as a vehicle con- cells were incubated only with DMSO not containing 15 trol, and the group indicated as "SiN Zalto" is a con- GW9662, and then zaltoprofen was added. The left- trol group for which the cells were infected with neg- most bars represent results of a group for which the ative-siRNA, and then cultured with zaltoprofen. cells were cultured without any drug, and the second [Fig. 71] Graphs showing results of suppression of bars from the left represent results of a group for proliferation of PVNS cells cultured in a zaltoprofen which the cells were incubated with mM 1 of 20 orrosiglitazone-containing medium. The experimen- GW9662, and then DMSO not containing zaltopro- tal groups are shown under the horizontal axes, and fen was added. the vertical axes represent the live cell count meas- [Fig. 69] A graph showing results of suppression of ured on the basis of absorbance at 450 nm using proliferation of cultured cells of chondrosarcoma CCK-8 (Dojindo). The left graph shows results of the (SW1353), which were cultured in a zaltoprofen or 25 groups for which the cells were infected with a neg- rosiglitazone-containing medium. The experimental ative siRNA designed so as not to suppress gene groups are shown under the horizontal axis, and the expression. The right graph shows results of the vertical axis represent live cell count measured on groups for which the cells were infected with PPAR γ- the basis of absorbance at 450 nm using CCK-8 siRNA designed so as to suppress PPAR Y gene ex- (Dojindo). From the left, the group indicated as "Si 30 pression. The groups of which results are shown in Control" is a group for which PPAR γgene expression the left graph are, from the left, a group for which the was suppressed with PPARγ-siRNA, and then the cells were infected with a negative siRNA, and then cells were cultured with DMSO as a vehicle control, cultured with DMSO as a vehicle control, a group for the group indicated as "Si Zalto" is a group for which which the cells were infected with a negative siRNA, PPARγ gene expression was suppressed with35 and then cultured with 100 mM rosiglitazone, and a PPARγ-siRNA, and then the cells were cultured with group for which the cells were infected with a nega- zaltoprofen, the group indicated as "SiN Control" is tive siRNA, and then cultured with 400 mM zaltopro- a control group for which the cells were infected with fen. The groups of which results are shown in the negative-siRNA, and then cultured with DMSO as a right graph are, from the left, a group for which the vehicle control, the group indicated as "SiN Zalto" is 40 cells were infected with PPAR γ-siRNA, and then cul- a control group for which the cells were infected with tured with DMSO as a vehicle control, a group for negative-siRNA, and then cultured with zaltoprofen, which the cells were infected with PPARγ-siRNA, the group indicated as "Si Control" is a group for and then cultured with 100 mM rosiglitazone, and a which PPAR Y gene expression was suppressed with group for which the cells were infected with PPARγ - PPARγ-siRNA, and then the cells were cultured with 45 siRNA, and then cultured with 400 mM zaltoprofen. DMSO as a vehicle control, the group indicated as [Fig. 72] A Graph showing results of suppression of "Si Rosi" is a group for which PPARγ gene expres- proliferation of cultured cells of bone sarcoma (HOS) sion was suppressed with PPARγ-siRNA, and then cultured in a zaltoprofen-containing medium. The cultured with rosiglitazone, the group indicated as horizontal axis represents the concentration of the "SiN Control" is a control group for which the cells 50 drug, and the vertical axis represents the relative were infected with negative-siRNA, and then cul- number of migrated cells. tured with DMSO as a vehicle control, and the group [Fig. 73] Graphs showing results of suppression of indicated as "SiN Rosi" is a control group for which cell migration of cultured cells of chondrosarcoma the cells were infected with negative-siRNA, and (HOS) cultured in an acetaminophen, celecoxib, in- then cultured with rosiglitazone. 55 domethacin, or diclofenac-containing medium. The [Fig. 70] A graph showing results of suppression of concentrations indicated under the horizontal axes proliferation of cultured cells of chondrosarcoma (H- are the concentrations of the drugs, and the vertical EMC-SS), which were cultured in a zaltoprofen-con- axes represent the relative number of migrated cells.

19 37 EP 3 050 573 A1 38

The groups indicated as Control are vehicle control are shown photographs of the cells that invaded un- groups for which DMSO not containing each drug der the condition of adding each drug. They are pho- was added. The added drugs are shown at the tops tographs of the cells that passed through a mem- of the graphs, respectively. brane coated with Matrigel under the conditions of [Fig. 74] Graphs showing results of suppression of 5 adding zaltoprofen (200 mM, 300 mM, 400 mM), tro- cell migration of cultured cells of chondrosarcoma glitazone (50 mM, 100 mM), or rosiglitazone (100 mM, (SW1353) cultured in a rosiglitazone, troglitazone, 200 mM). "Control" represents a vehicle control for zaltoprofen, or pioglitazone-containing medium. The which only DMSO was added in the same volume concentrations indicated under the horizontal axes as that used for addition of the drugs. Under the con- are the concentrations of the drugs, and the vertical 10 ditionthat 400 mM zaltoprofenor 100 mMtroglitazone axes represent the relative numbers of migrated was added, there were cells in such a number that cells. The groups indicated as Control are vehicle they only sparsely existed, and they rolled into small control groups for which DMSO not containing each balls. Therefore it can be seen that not only the sup- drug was added. The added drugs are shown at the pression of cell invasion, but also cell injury was re- tops of the graphs, respectively. 15 alized. [Fig. 75] A graph showing results of suppression of [Fig. 78] A graph showing results of suppression of cell invasion of cultured cells of chondrosarcoma cell invasion of cultured cells of chondrosarcoma (SW1353) cultured in a troglitazone, pioglitazone, (SW1353) cultured in a zaltoprofen-containing me- zaltoprofen, diclofenac, rosiglitazone, acetami- dium. Settings of the drugs are shown under the hor- nophen, indomethacin, or celecoxib-containing me- 20 izontal axis, and the vertical axis represents the rel- dium. Types and concentrations of the drugs are ative number of invaded cells. The black bars rep- shown under the horizontal axis, and the vertical axis resent the relative numbers of invaded cells obtained represents the relative number of invaded cells. The after the cells were incubated beforehand with 1 mM group indicated as Control is a vehicle control group (final concentration) of GW9662, which is an irre- for which DMSO not containing any drug was added. 25 versible antagonist and then zaltoprofen was added. [Fig.76] Photographs showing culturedcells of chon- The white bars represent the relative numbers of in- drosarcoma (SW1353) causing cell invasion, which vaded cells obtained after the cells were incubated were cultured in an acetaminophen, pioglitazone, in- only with DMSO not containing GW9662, and then domethacin, diclofenac, or celecoxib-containing me- zaltoprofen was added. The leftmost bars represent dium. The group indicated as Control is a vehicle 30 results of a group for which the cells were cultured control group for which DMSO not containing any without any drug, the second bars from the left rep- drug was added (see Example 24). For analysis of resent results of a group for which the cells were the cell invasion, Matrigel™ Invasion Chamber (BD incubated with 1 mM GW9662, and then 200 mM zal- Bioscience, catalog number 354480) using Matrigel toprofen was added, the third bars from the left rep- as a matrix was used. There are shown photographs 35 resent results of a group for which the cells were of the cells that invaded under the condition of adding incubated with 1 mM GW9662, and then 300 mM zal- each drug. They are photographs of the cells that toprofen was added, and the rightmost bars repre- passed through a membrane coated with Matrigel sent results of a group for which the cells were incu- under the conditions that acetaminophen (200 mM), bated with 1 mM GW9662, and then 400 mM zalto- pioglitazone (100 mM), indomethacin (200 mM), di- 40 profen was added. clofenac (200 mM), or celecoxib (50 mM) was added. [Fig. 79] A table summarizing cases of giant cell tu- "Control" represents a vehicle control for which only mor of bone of patients who took zaltoprofen (So- DMSO was added in the same volume as that used leton Tablet (registered trademark)). There are for addition of each drug. Under the condition of add- shown age, sex, occurring part of giant cell tumor of ing 50 mM celecoxib, there were cells in such a45 bone, differentiation of incipience or recurrence, fol- number that they only sparsely existed, and they low-up period, response rate, postoperative period, rolled into small balls. Therefore, it can be seen that treatment/drug exposure period, presence or ab- not only the suppression of cell invasion, but also sence of postoperative recurrence, and comment for cell injury was realized. each case, as well as number of drawing showing [Fig.77] Photographs showing culturedcells of chon- 50 photograph of affected part for the cases for which drosarcoma (SW1353) causing cell invasion, which such a photograph is shown. The symbol "PR" in the were cultured in a zaltoprofen, troglitazone, or ros- column of response rate means "partial response", iglitazone-containing medium. The group indicated "SD" means "stable disease", and "PD" means "pro- as Control is a vehicle control group for which DMSO gressive disease". Zometa (registered trademark) not containing any drug was added (see Example 55 mentioned in the column of comment is zoledronic 24). For analysis of the cell invasion, Matrigel™ In- acid hydrate Injection, and "Denosumab" is "anti- vasion Chamber (BD Bioscience, catalog number RANKL human monoclonal antibody (trade name, 354480) using Matrigel as a matrix was used. There Ranmark)".

20 39 EP 3 050 573 A1 40

[Fig. 80] A graph showing shrinking ratios of giant zaltoprofen over about 35 weeks, the diameter of the cell tumor of bone of giant cell tumor of bone patients giant cell tumor of bone that metastasized to the lung who took zaltoprofen (Soleton Tablet (registered shrank from 8.2 mm to 7.9 mm. trademark)). The vertical axis represents shrinking [Fig. 84] A table showing evaluation criteria of ratio. A shrinking ratio of 70% means that the giant 5 Karnofsky Performance Status (KPS). KPS is an cell tumor of bone shrank to a size of 30% of the size evaluation method for classifying patient’s condi- observed before taking zaltoprofen, and a shrinking tions into ten stages of 100 to 0 according to the ratio of -20% means that the giant cell tumor of bone criteria shown in Fig. 84, and a higher score means grew to a size of 120% of the size observed before better performance of the patient for everyday activ- taking zaltoprofen. The alphabets mentioned under 10 ities. the horizontal axis are alphabets for specifying the [Fig. 85] A table summarizing effects of continuous cases (see Fig. 79). In this specification, the shrink- taking of zaltoprofen (Soleton Tablet (registered ing ratio means a ratio of shrinkage of tumor ob- trademark)) on giant cell tumor of bone. There are served after continuous administration of zaltopro- mentioned KPS determined before the start of taking fen, based on the size of the tumor observed before 15 zaltoprofen and after continuous taking of zaltopro- the administration of zaltoprofen. More specifically, fen, presence or absence of osteosclerosis deter- inthis specification, the shrinking ratiomeans avalue mined by radiographic examination, presence or ab- obtained by subtracting 100 from a value calculated sence of osteosclerosis determined by CT, and by dividing a length for one direction of tumor in the shrinking ratio of giant cell tumor of bone for each maximum cut surface thereof on an MRI image or 20 case. X-ray CT image observed after continuous adminis- [Fig. 86] A table summarizing cases of PVNS of pa- tration of zaltoprofen as the numerator with the cor- tients who took zaltoprofen (Soleton Tablet (regis- responding length on such an image as mentioned tered trademark)). There are shown age, sex, occur- above observed before continuous administration of ring part of PVNS, differentiation of incipience or re- zaltoprofen as the denominator, and represented in 25 currence, treatment period, drug exposure period, terms of percentage. For example, if a length of tu- response rate, and comment for each case, as well mor for one direction in the maximum cut surface as number of drawing showing photograph of affect- thereof of 50 mm observed before administration of ed part for the cases for which such a photograph is zaltoprofen shrinks to 35 mm after the administration shown. The symbol "PR" in the column of response of zaltoprofen, the shrinking ratio is 30%. 30 rate means "partial response", "SD" means "stable [Fig. 81] Photographs showing transversal MRI im- disease", and "PD" means "progressive disease". ages of affected part (proximal part of left fibula) of [Fig. 87] A graph showing shrinking ratios of PVNS a giant cell tumor of bone patient (case c) who took of PVNS patients who took zaltoprofen (Soleton Tab- zaltoprofen (Soleton Tablet (registered trademark)), let (registered trademark)). The vertical axis repre- which photographs were obtained before (Decem- 35 sents the shrinking ratio. A shrinking ratio of 70% ber 13, 2012) and after (February 13, 2013) taking means that the giant cell tumor of bone shrank to a zaltoprofen. It can be seen that, as a result of taking size of 30% of the size observed before taking zal- zaltoprofen over about nine weeks, the diameter of toprofen, and a shrinking ratio of -10% means that the giant cell tumor of bone shrank from 23.3 mm to the giant cell tumor of bone grew to a size of 110% 21.0 mm. 40 of the size observed before taking zaltoprofen. The [Fig. 82] Photographs showing frontal and transver- alphabets mentioned under the horizontal axis are sal MRI images of affected part (distal part of right alphabets for specifying the cases (see Fig. 86). tibia) of a giant cell tumor of bone patient (case d) [Fig. 88] Photographs showing frontal MRI images who took zaltoprofen (Soleton Tablet (registered of affected part (shoulder joint) of a PVNS patient trademark)), which photographs were obtained be- 45 (case a) who took zaltoprofen (Soleton Tablet (reg- fore (July 4, 2013) administration of zaltoprofen, and istered trademark)), which photographs were ob- X-ray CT images of the same obtained after the ad- tained before (September 6, 2012) and after (August ministration (August 29, 2013). It can be seen that, 20, 2013) administration of zaltoprofen. It can be as a result of taking zaltoprofen over about eight seen that, as a result of taking zaltoprofen over about weeks, the diameter of the giant cell tumor of bone 50 50 weeks, the diameter of PVNS shrank from 27.7 shrank from 21.7 mm to 20.5 mm. mm to 7.9 mm. [Fig. 83] Photographs showing transversal X-ray CT [Fig. 89] Photographs showing sagittal MRI images images of affected part (lung metastasis part) of a of affected part (right knee joint) of a PVNS patient giant cell tumor of bone patient (case e) who took (case c) who took zaltoprofen (Soleton Tablet (reg- zaltoprofen (Soleton Tablet (registered trademark)), 55 istered trademark)), which photographs were ob- which photographs were obtained before (January tained before (December 6, 2012) and after (August 17, 2013) and after (September 19, 2013) taking zal- 1, 2013) administration of zaltoprofen. It can be seen toprofen. It can be seen that, as a result of taking that, as a result of taking zaltoprofen over about 35

21 41 EP 3 050 573 A1 42

weeks, the major axis of PVNS shrank from 59.5 mm is not particularly limited, so far as giant cells developed to 47.2 mm. in a bone and soft tissue are observed in the tumor. In [Fig. 90] A table summarizing effects of continuous particular, the present invention can be preferably ap- administration of zaltoprofen (Soleton Tablet (regis- plied to a tumor that generates giant cells in circumfer- tered trademark)) on PVNS. There are mentioned 5 ences of bone, joint, or tendon sheath. The tumor in which KPS determined before the start of taking zaltopro- giant cells developed in a bone and soft tissue are ob- fen and after continuous taking of zaltoprofen, and served is preferably a benign tumor. Examples of such shrinking ratio of giant cell tumor of bone for each a tumor include, for example, giant cell tumor of bone case. (GCTB), giant cell tumor of tendon sheath (GCTT), pig- 10 mented villonodular synovitis (PVNS), and the like. Ex- Modes for Carrying out the Invention amples of benign giant cell tumor occurring in a bone and soft tissue also include chondroblastoma, nonossifying [0073] The present invention can be carried out by pro- fibroma, osteoblastoma, aneurysmal bone cyst, and the ducing a drug containing a substance having a PPARγ- like. agonistic activity and/or a PPARγ expression-inducing 15 [0079] Examples of the chondrosarcoma to which the activity as an active ingredient for the purpose of thera- present invention can be applied include conventional peutic treatment of a patient suffering from giant cell tu- chondrosarcoma, periosteal chondrosarcoma, mesen- moroccurring in abone and softtissue, chondrosarcoma, chymal chondrosarcoma, dedifferentiated chondrosar- or bone sarcoma, prophylactic treatment for such a dis- coma, clear-cell chondrosarcoma, extraskeletal myxoid ease, or prevention of metastasis in such a patient as 20 chondrosarcoma, and the like. mentioned above. [0080] Examples of the bone sarcoma to which the [0074] The present invention can also be carried out present invention can be applied include those of oste- by administering a drug containing a substance having oblast type called conventional type, chondroblast type, a PPARγ-agonistic activity and/or a PPARγ expression- fibroblast type, vasodilatation type, small cellular type, inducing activity as an active ingredient to a patient suf- 25 parosteal bone sarcoma, and the like. fering from giant cell tumor occurring in a bone and soft [0081] The present invention can also be applied to a tissue, chondrosarcoma, or bone sarcoma. tumor in which expression of PPAR Y is observed. Exam- [0075] The object to which the present invention can ples of such a tumor in which expression of PPARy is be applied is not particularly limited, so long as a verte- observed include breast cancer, colon cancer, lung can- brate is chosen as the object, and the object is preferably 30 cer, thyroid gland cancer, esophageal cancer, gastric a mammal, more preferably human, ape, canine, feline, cancer, pancreatic cancer, liver cancer, kidney cancer, bovine, equine, swine, ovine, caprine, or lagomorph, vesical cancer, ovarian cancer, uterine cervix carcinoma, most preferably human. prostate cancer, malignant melanoma, leukemia, malig- [0076] When the object to which the present invention nant lymphoma, liposarcoma, leiomyosarcoma, bone can be applied is human, the present invention can be 35 sarcoma, and the like. applied not only to a patient who has been clinically di- [0082] PPARY is a transcriptional factor protein be- agnosed to have giant cell tumor occurring in a bone and longing to the intranuclear receptor superfamily, and a soft tissue, chondrosarcoma, or bone sarcoma, but also substance having a PPARγ-agonistic activity and/or a to a person who is suspected to have, or predicted to PPARY expression-inducing activity can induce apopto- 40 develop such a disease in future. The present invention sis or fat cell differentiation mediated by PPAR Y in giant can also be applied to a patient suffering from metastasis cell tumors occurring in a bone and soft tissue, chondro- of cells of giant cell tumor occurring in a bone and soft sarcoma, or bone sarcoma. tissue, chondrosarcoma, or bone sarcoma as a primary [0083] Therefore, in the present invention, as a sub- disease. stance that can be used as the agent for prophylactic [0077] Embodiments of the agent for prophylactic45 treatment, therapeutic treatment, or prevention of metas- treatment, therapeutic treatment, or prevention of metas- tasis of giant cell tumor occurring in bone and soft tissue, tasis of the present invention include those for use in chondrosarcoma, or bone sarcoma, a substance having restoration or formation of a bone in a patient suffering a PPARγ expression-inducing activity and/or a PPARγ- from giant cell tumor occurring in a bone and soft tissue, agonistic activity is desirable. chondrosarcoma, or bone sarcoma. Embodiments of the 50 [0084] In the present invention, the PPARγ-agonistic agent for prophylactic treatment, therapeutic treatment, activity means an activity for binding to PPAR γ to promote or prevention of metastasis of the present invention also transcription of a gene existing downstream from the include those for use in improving ability to carry out eve- PPAR response element (PPRE). Any substance having ryday activities of a patient suffering from giant cell tumor this activity can be regarded as a PPAR Y agonist. Exam- occurring in a bone and soft tissue, chondrosarcoma, or 55 ples of gene existing downstream from the PPAR re- bone sarcoma. sponse element (PPRE) include an apoptosis-related [0078] The giant cell tumor occurring in a bone and gene, fat cell differentiation-related gene, arteriosclero- soft tissue to which the present invention can be applied sis-related gene, and anti-inflammation-related gene.

22 43 EP 3 050 573 A1 44

[0085] However, in the present invention, PPARY ag- nesil, celecoxib, etodolac, meloxicam, mofezolac, and onist refers to a substance that can bind to PPARY to acemetacin. promote transcription of a gene existing downstream [0095] In the present invention, the thiazolidinedione from the PPAR response element (PPRE), or a sub- derivative is not particularly limited so long as it is a sub- stance selected from the group consisting of a non-ster- 5 stance having the thiazolidinedione structure and having oidal anti-inflammatory agent, a thiazolidinedione deriv- a PPARγ-agonistic activity. ative, an angiotensin II receptor antagonist having a [0096] Examples of the thiazolidinedione derivative in- PPARγ-agonistic activity, and an endogenous PPARγ clude pioglitazone, rosiglitazone, troglitazone, isaglita- agonist. zone, netoglitazone, rivoglitazone, balaglitazone, lobeg- 10 [0086] In the present invention, the PPARY agonists litazone, englitazone, ciglitazone, and the like. exemplified below include pharmacologically acceptable [0097] In the present invention, the angiotensin II re- salts, solvates, tautomers, and stereoisomers thereof in ceptor antagonist having a PPARγ-agonistic activity is addition to the exemplified substances themselves, even not particularly limited so long as it is a substance having if there are not explicitly indicated as "salt", "solvate", an angiotensin II receptor antagonistic activity and an "tautomer", "stereoisomer", and the like 15 activity as a PPAR γ agonist. As the angiotensin II recep- [0087] In the present invention, substance having a tor antagonist having a PPARγ-agonistic activity, irbe-

PPARY expression-inducing activity refers to a sub- sartan and telmisartan are especially preferred. stance that can promote transcription of a PPARY gene [0098] In the present invention, the endogenous from a genome gene. PPARγ agonist is not particularly limited so long as it is [0088] In the present invention, as a substance having 20 a substance endogenously possessed by an organism a PPARY expression-inducing activity, non-steroidal an- in the organism’s own body, and is a substance having ti-inflammatory agents can be mentioned. an activity as a PPARγ agonist. As the endogenous [0089] Non-steroidal anti-inflammatory agents have a PPARγ agonist, 15-deoxy-Δ12,14-prostagladin J2, 15- PPARγ expression-inducing activity, and a PPARγ-ago- hydroxyeicosatetraenoic acid, 9-hydroxyoctadecadieno- nistic activity. 25 ic acid, 13-hydroxyoctadecadienoic acid, nitrolinoleic ac- [0090] In the present invention, the non-steroidal anti- id, or a long chain fatty acid is preferred. inflammatory agent means a non-steroidal anti-inflam- [0099] The response rate used as an index of effect of matory agent in a general meaning, and it is not partic- an anticancer agent is a ratio representing effectiveness ularly limited so long as it is a substance having anti- of drug therapy such as those using anticancer agent. It inflammatory activity, analgesic action, and antipyretic 30 means the total of the ratio of "complete response (CR)", action based on inhibition of cyclooxygenase 1 and/or which means complete disappearance of tumor, and the cyclooxygenase 2. ratio of "partial response (PR)", which means 30% or [0091] Examples of the non-steroidal anti-inflammato- more of shrinkage of tumor, determined according to the ry agent include, for example, zaltoprofen, diclofenac, general standards (RECIST) using diagnostic imaging indomethacin, proglumetacin, indometacin farnesil,35 such as CT for an evaluation object. The "complete re- celecoxib, etodolac, meloxicam, mofezolac, acemetacin, sponse (CR)" mentioned above means a state that tumor oxaprozin, acetaminophen, lornoxicam, ampiroxicam, has completely disappeared, and the "partial response piroxicam, naproxen, loxoprofen, rofecoxib, ethenza- (PR)" means a state that the total of sizes of tumor has mide, diflunisal, aluminoprofen, nabumetone, ketopro- decreased by 30% or more. "Stable disease (SD)" means fen, acetylsalicylic acid, ibuprofen, pranoprofen, and40 a state that size of tumor has not changed, and "progres- sulindac. sive disease (PD)" means a state that the total of sizes [0092] Among them, non-steroidal anti-inflammatory of tumor has increased by 20% or more, and increased agents especially preferred for the present invention are by 5 mm or more in terms of absolute value, or a state zaltoprofen, diclofenac, indomethacin, proglumetacin, that new lesion has appeared. Although it is desirable for indometacin farnesil, celecoxib, etodolac, meloxicam, 45 anticancer agents to provide complete response or par- mofezolac, acemetacin, oxaprozin, acetaminophen, lor- tial response, if the ability to carry out everyday activities noxicam, ampiroxicam, piroxicam, naproxen, loxopro- is maintained, the therapeutic effect can be regarded de- fen, rofecoxib, ethenzamide, diflunisal, aluminoprofen, sirable, even if the disease is determined to be "stable and nabumetone. disease" or "progressive disease". [0093] For the present invention, more preferred non- 50 [0100] In the present invention, the ability to carry out steroidal anti-inflammatory agents are zaltoprofen, di- everyday activities can be determined according to, for clofenac, indomethacin, proglumetacin, indometacin far- example, the Karnofsky performance status (henceforth nesil, celecoxib, etodolac, meloxicam, mofezolac, referred to as "KPS"). The score of KPS ranges from zero acemetacin, oxaprozin, acetaminophen, lornoxicam, point to 100 points, and a higher score means better per- ampiroxicam, piroxicam, and naproxen. 55 formance of the patient for everyday activities. KPS is [0094] For the present invention, most preferred non- used for judging prognosis of patient, measuring change steroidal anti-inflammatory agents are zaltoprofen, di- of activity ability, determining whether patient can partic- clofenac, indomethacin, proglumetacin, indometacin far- ipate in a clinical trial, and the like. KPS is also useful as

23 45 EP 3 050 573 A1 46 one of indices for determining quality of life. lubricant, disintegrating aid, oxidation inhibitor or antioxi- [0101] The dose of the agent for prophylactic treat- dant, emulsifier, dispersing agent, suspending agent, ment, therapeutic treatment, or prevention of metastasis dissolving agent, dissolving aid, thickener, pH adjustor of the present invention can be appropriately chosen de- or buffering agent, stabilizer, antiseptic or preservative, pending on type of active ingredient, object of adminis- 5 bacteriocide or antibacterial agent, antistatic agent, cor- tration, age, body weight, sex, and conditions (general rigent or masking agent, colorant, odor-masking agent condition, pathological condition, presence or absence or perfume, refrigerant, antifoam, isotonic agent, sooth- of complication, and the like) of the object of administra- ing agent, and the like can be used. These additives can tion, administration period, dosage form, administration be used independently or in a combination of two or more method, and the like. For example, the dose of zaltopro- 10 kinds of them. fen for oral administration to a human adult is preferably [0105] For the agent for prophylactic treatment, thera- 80 to 1200 mg/day, more preferably 160 to 960 mg/day, peutic treatment, or prevention of metastasis of the further preferably 240 to 720 mg/day, most preferably present invention, a carrier or excipient chosen as re- 480 mg/day. quired depending on administration route and use of [0102] The agent for prophylactic treatment, therapeu- 15 preparation from the ingredients (for example, excipi- tic treatment, or prevention of metastasis of the present ents, binders, disintegrating agents, lubricants, coating invention can be prepared by conventional methods for agents, and the like) described in, for example, besides producing pharmaceutical preparations, for example, the Japanese Pharmacopoeia, (1) Handbook of Pharmaceu- production methods described in Japanese Pharmaco- tical Additives, Maruzen Co., Ltd., 1989, (2) "Encyclope- poeia 16th Edition, or similar methods, using, besides 20 dia of Pharmaceutical Additive 2007" (Yakuji Nippo, pub- the active ingredient, carrier component, additives, and lished on July, 2007), (3) Pharmaceutics, 5th revised edi- the like, as required. tion, Nankodo Co., Ltd., 1997, (4) Japan Pharmaceutical [0103] The administration method of the agent for pro- Excipient Standards 2003 (Yakuji Nippo, August, 2003), phylactic treatment, therapeutic treatment, or prevention and the like can be used. of metastasis of the present invention may be oral ad- 25 [0106] As the carrier or excipient used for such phar- ministration, or parenteral administration. Examples of maceutical preparations, for example, saccharides or the parenteral administration include, for example, intra- sugar alcohols such as lactose, glucose, sucrose, man- muscular administration, enteral administration, trans- nitol, sorbitol and xylitol, starches such as potato starch mucosal administration, transpulmonary administration, and corn starch, calcium carbonate, calcium phosphate, dermal administration, transnasal administration, vaginal 30 calcium sulfate, polysaccharides such as crystalline cel- administration, intraoral administration, epidural admin- lulose (including microcrystalline cellulose), silicon oxide istration, intravenous administration, intrathecal admin- or silicate such as light anhydrous silicic acid, glycyr- istration, sublingual administration, rectum administra- rhizae radix pulverata, gentianae radix pulverata, and the tion, instillation administration, intraarterial administra- like can be used. tion, intraurethral administration, subcutaneous admin- 35 [0107] As the binder used for the pharmaceutical prep- istration, intracutaneous administration, and intraperito- arations, for example, gelatin, soluble starches such as neal administration. Examples of the dosage form for pregelatinized starch and partially pregelatinized starch, parenteral administration include injection (solution, gum arabic, tragacanth gum, polysaccharides such as lyophilized preparation, suspension, and the like), sup- dextrin and sodium arginate; synthetic polymers such as pository (anus suppository, vaginal suppository, and the 40 polyvinylpyrrolidone (PVP), polyvinyl ether, polyvinyl al- like), liquid for external use (infusion, poultice, aerosol, cohol (PVA), carboxyvinyl polymer, polyacrylic polymer, and the like), inhalant, patch, percutaneous absorption polylactic acid, and polyethylene glycol; cellulose ethers tape, cataplasm, skin external preparation, cream, gel, such as methylcellulose (MC), ethylcellulose (EC), car- ointment (dermatologic paste, liniment, lotion, and the boxymethylcellulose (CMC), carboxymethylcellulose so- like), and the like. As the dosage form for oral adminis- 45 dium, hydroxyethylcellulose (HEC), hydroxypropylcellu- tration, for example, gummi, syrup, jelly, chewable tablet, lose (HPC), and hydroxypropylmethylcellulose (HPMC), troche, dry syrup, buccal tablet, film-coated tablet, film and the like can be used. preparation, pill, solution or suspension for oral adminis- [0108] As the disintegrating agent, sodium arginate, tration, oral disintegrating tablet, hard capsule, subtilized carboxymethyl starch sodium, carmellose, carmellose granule, powder, sublingual tablet, uncoated tablet, en- 50 calcium, carmellose sodium, croscarmellose sodium, teric coated tablet, sugar-coated tablet, soft capsule, crospovidone, gelatin powder, starch, agar, crystalline emulsion, adhesive tablet, powder, granule, and the like cellulose, calcium carbonate, sodium hydrogencar- can be used. bonate, low-substituted hydroxypropylcellulose, and the [0104] In the preparation of the agent for prophylactic like can be used. treatment, therapeutic treatment, or prevention of metas- 55 [0109] As the lubricant used for the pharmaceutical tasis of the present invention, known additives can be preparations, for example, magnesium stearate, talc, hy- used as required according to administration route, dos- drogenated vegetable oil, Macrogoal, and the like can be age form, and the like. As such additives, for example, used.

24 47 EP 3 050 573 A1 48

[0110] When an injection is prepared, it can be pre- of gelatin sponge and polyvinyl alcohol are clinically pared with adding a pH adjuster, buffering agent, stabi- used. lizer, solubilizer, and the like as required. [0116] Gelatin sponge consists of gelatin formed in a [0111] When tablets or granules are prepared, as coat- spongy shape, and is also known as Spongel (registered ing agent, for example, saccharides, hydroxypropylcel- 5 trademark) or Gelfoam (registered trademark). If a thin lulose, sorbitol, purified shellac, gelatin, cellulose deriv- strip ofgelatin sponge is injectedinto an artery,the gelatin atives such as ethylcellulose, hydroxypropylmethylcellu- sponge stagnates in the objective artery, and embolizes lose, and hydroxymethylcellulose, polyoxyethylene gly- only the artery for a certain period of time together with col, cellulose acetate phthalate, hydroxypropylmethyl- thrombus formed there. The gelatin sponge strip integrat- cellulose phthalate, methyl methacrylate/(meth)acrylate 10 ed with the thrombus is gradually absorbed into the body copolymer, Eudragit (methacrylic acid/acrylic acid copol- on that spot, the artery is recanalized in about one or two ymer), and the like can be used. The coating agent may weeks, and the gelatin sponge disappears from the inside be an enteric component such as cellulose phthalate, of the body in one month. hydroxypropylmethylcellulose phthalate, and methyl [0117] A substance in the form of a fine strip of a size methacrylate/(meth)acrylic acid copolymer, or gastric 15 of about 100 to 300 mm, 300 to 500 mm, or 500 to 700 soluble component consisting of a polymer containing a mm consisting of polyvinyl alcohol is a local infusion for basic component such as dialkylaminoalkyl (meth)acr- artery embolization that eternally remains in an embol- ylate (Eudragit and the like). The pharmaceutical prepa- ized blood vessel, and consists of a spherical substance ration may also be a capsule of which capsule itself con- of the standardized size, and therefore it does not clog tains these enteric components and gastric soluble com- 20 a catheter, and is easily used. ponents. [0118] Various spherical particles for artery emboliza- [0112] The present invention can also be implemented tion have been developed, and spherical PVA (Bead by injecting or embedding a local infusion for artery em- Block, Biocompatibles), non-water-absorptive spherical bolization or artificial bone containing a substance having particles of acrylic copolymer impregnated and coated a PPARγ-agonistic activity and/or a PPARγ expression- 25 with pig gelatin (Embosphere, Biosphere Medical), inducing activity as an active ingredient into an artery of spherical PVA imparted with drug dissolution ability (DC- affected part at the time of surgical operation of a patient Bead, Biocompatibles), water-absorptive and swellable suffering from giant cell tumor occurring in a bone and bead consisting of polyvinyl alcohol/acrylic acid copoly- soft tissue, chondrosarcoma, or bone sarcoma. mer (Hepasphere, Biosphere Medical), acrylic hydrogel [0113] A substance having a PPARγ -agonistic activity 30 having special fluorine coating (Embozene, CeloNova), and/or a PPAR γ expression-inducing activity can be used and the like are clinically used. in a state that it is contained in a local infusion for artery [0119] By preliminarily adding a substance having a embolization or artificial bone. By injecting or embedding PPARγ-agonistic activity and/or a PPAR γ expression-in- a local infusion for artery embolization or artificial bone ducing activity to such a local infusion for artery emboli- containing a substance having a PPAR γ-agonistic activ- 35 zation as mentioned above, not only giant cell tumor or ity and/or a PPARγ expression-inducing activity as an chondrosarcoma can be annihilated by stopping delivery active ingredient into an artery of affected part at the time of nutrition and oxygen to the giant cell tumor or chond- of surgical operation of a patient suffering from giant cell rosarcoma, but also apoptosis can be induced in the giant tumor occurring in a bone and soft tissue, chondrosar- cell tumor or chondrosarcoma to make the giant cell tu- coma, or bone sarcoma, apoptosis can be induced in the 40 mor or chondrosarcoma disappear, and the tumor can giant cell tumor or chondrosarcoma to make the tumor be differentiated into fat cells and thereby made to dis- disappear, or the tumor can be differentiated into fat cells, appear. Therefore, a higher curative effect can be ob- and thereby made to disappear. Furthermore, by using tained. the local infusion for artery embolization or artificial bone [0120] Further,it is also frequently performed to, before containing a substance having a PPAR γ-agonistic activ- 45 the artery embolization, inject iodinated poppy seed oil ity and/or a PPARγ expression-inducing activity as an fatty acid ethyl esters containing an anticancer agent into active ingredient, recurrence and/or metastasis of giant a tumor through a catheter inserted up to a position just cell tumor occurring in a bone and soft tissue, chondro- before the tumor. Since iodinated poppy seed oil fatty sarcoma, or bone sarcoma can be prevented. acid ethyl esters constitute an oily contrast medium, the [0114] The artery embolization is a therapy for annihi- 50 anticancer agent can be delivered into the inside of the lating a tumor by injecting a substance that embolizes an tumor cells with confirming the injection state of the an- artery upstream of giant cell tumor or chondrosarcoma ticancer agent. Therefore, by injecting iodinated poppy so that nutrition and oxygen are not delivered to the giant seed oil fatty acid ethyl esters containing a substance cell tumor or chondrosarcoma. having a PPARγ-agonistic activity and/or a PPARγ ex- [0115] Material of the local infusion for artery emboli- 55 pression-inducing activity into giant cell tumor or chond- zation is not particularly limited so long as a substance rosarcoma through a catheter inserted into an artery, and that can embolize arteries and adapts to living tissues is then performing artery embolization, a higher curative chosen, and various spherical particles including those effect can be obtained. Iodinated poppy seed oil fatty

25 49 EP 3 050 573 A1 50 acidethyl ester preparation iswell knownas Lipiodol (reg- [0127] Content of the substance having a PPARγ-ag- istered trademark). onistic activity and/or a PPARγ expression-inducing ac- [0121] In the present invention, the artificial bone tivity in the material of the artificial bone is preferably means an artificial material for compensating a defective about 0.1 to 25%, more preferably about 1 to 20%, still part of bone, and an artificial joint produced by using such 5 more preferably about 5 to 15%, of the weight of the ma- a material. The material of the artificial bone is not limited terial of the artificial bone. to non-biological materials, and includes biological ma- [0128] The screening method of the present invention terials. The biological materials include autologous bone, can be implemented with the following steps: homologous bone, and heterologous bone prepared through an artificial step such as adding a substance hav- 10 (1) the step of culturing a cell or tissue originating in ing a PPARγ-agonistic activity and/or a PPARγ expres- giant cell tumor occurring in a bone and soft tissue, sion-inducing activity. chondrosarcoma, or bone sarcoma in the presence [0122] When an ablative operation is performed as a or absence of a test substance, radical therapy for giant cell tumor occurring in a bone (2) the step of measuring one or more indices se- and soft tissue, chondrosarcoma, or bone sarcoma, it is 15 lected from the group consisting of those defined in necessary to fill up the defective part formed after curet- (a) to (g) mentioned below in the presence or ab- tage or resection of a lesion with an artificial bone to se- sence of the test substance; cure bone strength. Further, since giant cell tumor occur- ring in a bone and soft tissue, chondrosarcoma, or bone (a) one or more indices selected from the group sarcoma frequently occurs at a position near a joint, if an 20 consisting of gene expression amount of ablative operation is performed as a radical therapy, mo- PPARγ, and protein amount of PPARγ, tor functions must be recovered by using an artificial joint (b) one or more indices selected from the group after the curettage or resection of the lesion. consisting of gene expression amount of an ap- [0123] By using the artificial bone containing a sub- optosis-related gene, protein amount of a trans- stance having a PPAR γ-agonistic activityand/or a PPAR γ 25 lation product of an apoptosis-related gene, and expression-inducing activity as an active ingredient, re- biological activity of a translation product of an currence and/or metastasis of giant cell tumor occurring apoptosis-related gene, in a bone and soft tissue, chondrosarcoma, or bone sar- (c) one or more indices selected from the group coma can be prevented. consisting of gene expression amount of a fat [0124] In addition to strength, processability and com- 30 cell differentiation-related gene, protein amount patibility with bone are required for the material of artificial of a translation product of a fat cell differentia- bone, and as such a material of artificial bone, calcium tion-related gene, and biological activity of a phosphate type materials such as hydroxyapatite, metals translation product of a fat cell differentiation- such as titanium, titanium alloy, stainless steel, cobalt- related gene, chromium alloy, and tungsten, polymers such as poly- 35 (d) one or more indices selected from the group lactic acid, crosslinked polyethylene resin, silicon rubber, consisting of gene expression amount of an ar- Teflon (registered trademark), polyester, and PVA hy- teriosclerosis-related gene, protein amount of a drogel, glass, ceramics such as alumina and zirconia, translation product of an arteriosclerosis-related bone cement such as polymethyl methacrylate, proteins gene, and biological activity of a translation such as collagen and fibrin, polysaccharides such as chi- 40 product of an arteriosclerosis-related gene, tin and chitosan, coral materials, and composite materi- (e) one or more indices selected from the group als of these can be used. consisting of gene expression amount of an anti- [0125] The artificialbone canbe formed before surgical inflammation-related gene, protein amount of a operation by scraping a material, molding, three-dimen- translation product of an anti-inflammation-re- sional printing, or the like. As such artificial bone mate- 45 lated gene, and biological activity of a translation rials, calcium phosphate artificial bone materials similar product of an anti-inflammation-related gene, to the bone components are most abundant in types, and (f) an index consisting of a PPAR γ-agonistic ac- various kinds of such materials are commercially pro- tivity that can promote transcription of one or duced and clinically used, including Apacerum (regis- more kinds of genes selected from the group tered trademark), SuperPore (registered trademark), Bi- 50 consisting of an apoptosis-related gene, a fat opex (registered trademark), Bonetight (registered trade- cell differentiation-related gene, an arterioscle- mark), Bonefill (registered trademark), Neobone (regis- rosis-related gene, and an anti-inflammation-re- tered trademark), Regenos (registered trademark), OS- lated gene, ferion (registered trademark), and the like. (g) amount of lipid contained in a fat cell or fat [0126] The artificial joint is usually chosen from ready- 55 tissue, made products consisting of a combination of such ma- terials as metal, ceramics, and polyethylene resin, and and used by embedding and fixing it in a bone. (3) the step of selecting a test substance that chang-

26 51 EP 3 050 573 A1 52

es a value or values of the intracellular index or in- quence information is disclosed as GenBank accession dices in the presence of a test substance compared No. AAH06811. with the value or values of the intracellular index or [0136] Further, the PPARγ-agonistic activity can be indices observed in the absence of the test sub- confirmed by, for example, detecting dissociation of the stance. 5 co-repressor protein complex from the hetero-complex of PPARγ and RXR, or by detecting binding of the co- [0129] As for the preparation of cells originating in giant activator protein complex to the hetero-complex of cell tumor occurring in a bone and soft tissue, chondro- PPARγ and RXR. Furthermore, the PPAR γ-agonistic ac- sarcoma, or bone sarcoma used for the screening meth- tivity can also be confirmed by measuring expression lev- od of the present invention, those prepared by primarily 10 el of a downstream gene, or expression level of a lipid. culturing cells extracted by an ablative operation for tu- [0137] Further, amount of protein as the translation mor of a patient clinically diagnosed to have giant cell product of the apoptosis-related gene, fat cell differenti- tumor occurring in a bone and soft tissue, chondrosar- ation-related gene, arteriosclerosis-related gene, or anti- coma, or bone sarcoma, or cells of a cell line established inflammation-related gene can also be measured by a from such cells as mentioned above by a known method 15 known method on the basis of known information on each can be used. gene, as in the case of the measurement of amount of [0130] A cell line can be established according to a PPARγ protein already described. method ordinarily performed in this field, or according to [0138] As the method for measuring the PPARγ-ago- a description of published references. Further, the cells nistic activity, an ability to bind to PPARγ to change the derived from giant cell tumor occurring in a bone and soft 20 conformation of the PPAR γ protein can be measured, or tissue, chondrosarcoma, or bone sarcoma may consist amount of transcription product of a gene locating down- of an arbitrary tissue (for example, synovial membrane, stream from the PPAR response element (PPRE), or a joint, cartilage, and the like) containing the cells. translation product thereof can be measured. As for the [0131] Although the culture condition for the cells orig- method for measuring an ability to change the conforma- inating in giant cell tumor occurring in a bone and soft 25 tion of the PPAR γ protein, competitive binding observed tissue, chondrosarcoma, or bone sarcoma used for the for a known agonist may be used as an index, or surface screening method of the present invention can be appro- plasmon resonance may also be utilized. priately adjusted in accordance with a known method, [0139] Examples of the gene locating downstream specifically, and for example, they can be cultured ac- from the PPAR response element (PPRE) include, for cording to the methods described in the examples men- 30 example, an apoptosis-related gene, fat cell differentia- tioned in this specification. tion-related gene, arteriosclerosis-related gene, anti- in- [0132] In the present invention, the PPARγ-agonistic flammation-related gene, and the like. activity and PPARγ expression-inducing activity can be [0140] The apoptosis-related gene may be a gene car- confirmed by known methods. rying an apoptosis signal, or a marker gene for apoptosis. [0133] Specifically, expression amount of the PPARγ 35 Examples of the apoptosis-related gene include, for ex- gene can be measured by a method comprising extract- ample, those of caspase 3, p53, and the like. ing mRNAs from giant cell tumor occurring in a bone and [0141] As for the transcription product of caspase 3, soft tissue, chondrosarcoma, or bone sarcoma by a usual for example, nucleotide sequence information is dis- method, and performing a reverse transcription reaction closed as GenBank accession No. BC016926, and as and PCR using primers enabling amplification of the40 for the translation product thereof, amino acid sequence PPARγ transcription product. The transcription product information is disclosed as GenBank accession No. and translation product of PPAR γ are known, and for ex- AAH16926. The biological activity of the translation prod- ample, nucleotide sequence information of the transcrip- uct of the caspase 3 gene can be measured on the basis tion product is disclosed as GenBank accession No. of apoptosis, or can also be measured on the basis of BC006811. 45 the cysteine protease activity. [0134] Expression amount of each of the apoptosis- [0142] As for the transcription product of p53, for ex- related gene, fat cell differentiation-related gene, arteri- ample, nucleotide sequence information is disclosed as osclerosis-related gene, and anti-inflammation-related GenBank accession No. NM_000546, and as for the gene can also be measured by a known method on the translation product thereof, amino acid sequence infor- basis of known gene information, like the measurement 50 mation is disclosed as GenBank accession No. of expression amount of PPAR γ gene already described. NP_000537. The biological activity of the translation [0135] Further, amount of the PPARγ protein can be product of the p53 gene can be measured on the basis measured by, for example, a PPAR antigen-antibody re- of apoptosis, or can also be measured on the basis of action using an antibody directed to the PPARγ protein, change of control of cell cycle. and a fixed specimen or disrupted cells of giant cell tumor 55 [0143] The fat cell differentiation-related gene may be occurring in a bone and soft tissue, chondrosarcoma, or a gene that induces differentiation into fat cells, or may bone sarcoma as a measurement sample. As for the be a marker gene for fat cell differentiation. translation product of the PPARγ gene, amino acid se- [0144] Examples of the fat cell differentiation-related

27 53 EP 3 050 573 A1 54 gene include, for example, those of Setd8 (SET domain information is disclosed as GenBank accession No. containing (lysine methyltransferase) 8), Setdb 1 (SET NP_004788. The biological activity of the translation domain, bifurcated 1), LPL (Lipoprotein Lipase), leptin, product of the adiponectin gene can be measured on the FABP4/aP2 (fatty acid-binding protein-4), adiponectin, basis of the AMP kinase-activating action, fatty acid-com- a2Col6 (α chain 2 of type 6 collagen), and the like. 5 busting action, or saccharide incorporation-promoting [0145] As for the transcription product of Setd8, for ex- action exhibited when the product is allowed to act on ample, nucleotide sequence information is disclosed as cells of the liver or skeletal muscle. GenBank accession No. NM_020382, and as for the [0151] As for the transcription product of a2Col6, for translation product thereof, amino acid sequence infor- example, nucleotide sequence information is disclosed mation is disclosed as GenBank accession 10 No.as GenBank accession No. BC065509, and as for the NP_065115. The biological activity of the translation translation product thereof, amino acid sequence infor- product of the Setd8 gene can be measured on the basis mation is disclosed as GenBank accession No. of lipid production amount, or can also be measured on AAH65509. The biological activity of the translation prod- the basis of the enzyme activity for methylating the 20th uct of the a2Col6 gene can be measured on the basis of lysine of the histone H4 protein. 15 differentiation into fat cells as an index. [0146] As for the transcription product of Setdb1, for [0152] Examples of the arteriosclerosis-related gene example, nucleotide sequence information is disclosed include, for example, those of AT1R (angiotensin II re- as GenBank accession No. NM_012432, and as for the ceptor I), and the like. As for the transcription product of translation product thereof, amino acid sequence infor- AT1R, nucleotide sequence information is disclosed as mation is disclosed as GenBank accession 20 No.GenBank accession No. NM_000685, and as for the NP_036564. The biological activity of the translation translation product thereof, amino acid sequence infor- product of the Setdbl gene can be measured on the basis mation is disclosed as GenBank accession No. of lipid production amount, or can also be measured on NP_000676. The biological activity of the translation the basis of the enzyme activity for methylating the 9th product of the AT1R gene can be measured on the basis lysine of the histone H3 protein. 25 of detection of expression cells using angiotensin II as a [0147] As for the transcription product of LPL, for ex- ligand. ample, nucleotide sequence information is disclosed as [0153] Examplesof the anti-inflammation-related gene GenBank accession No. NM_000237, and as for the include, for example, those of NF-κB (nuclear factor ka- translation product thereof, amino acid sequence infor- ppa-light-chain-enhancer of activated B cells), and the mation is disclosed as GenBank accession 30 No.like. As for the transcription product of NF- κB, for exam- NP_000228. The biological activity of the translation ple, nucleotide sequence information is disclosed as product of the LPL gene can be measured on the basis GenBank accession No. NM_003998, and as for the of the lipoprotein lipase activity. translation product thereof, amino acid sequence infor- [0148] As for the transcription product of leptin, for ex- mation is disclosed as GenBank accession No. ample, nucleotide sequence information is disclosed as 35 NP_003989. The biological activity of the translation GenBank accession No. NM_000230, and as for the product of the NF- κB gene can be measured on the basis translation product thereof, amino acid sequence infor- of dissociation thereof from IκB, and can also be meas- mation is disclosed as GenBank accession No.ured on the basis of intranuclear transfer of NF- κB. NP_000221. The biological activity of the translation [0154] The lipid contained in fat cells or fat tissues is product of the leptin gene can be measured on the basis 40 not particularly limited, and examples include phosphol- of the appetite-suppressing activity observed when it is ipids, glycolipids, lipoproteins, acylglycerols, ceramides, intravenously administered to a mammal, or can also be and the like. measured on the basis of the intracellular signal ob- [0155] Specifically, as for the measurement of the served when the leptin protein binds to a cell that ex- PPARγ expression-inducing activity, it can be examined presses a leptin receptor. 45 by preparing an RNA (for example, total RNA, or mRNA) [0149] As for the transcription product of FABP4/aP2 fraction from a cell, and detecting the transcription prod- (fat cell-specific fatty acid-binding protein), for example, uct of the PPARγ gene contained in the fraction. Such nucleotide sequence information is disclosed as Gen- an RNA fraction can be prepared by a known method Bank accession No. NM_001442, and as for the trans- such as the guanidine-CsCl ultracentrifugation method lation product thereof, amino acid sequence information 50 and the AGPC method, and total RNA of high purity can is disclosedas GenBank accession No. NP_001433. The be quickly and conveniently prepared from a small biological activity of the translation product of the number of cells by using a commercial kit for RNA ex- FABP4/aP2 gene can be measured on the basis of effi- traction (for example, RNeasy Mini Kit produced by QIA- ciency of lipolysis of a cell that expresses the product. GEN and the like). Examples of the means for detecting [0150] As for the transcription product of adiponectin, 55 a transcription product of a gene in an RNA fraction in- for example, nucleotide sequence information is dis- clude, for example, a method of using hybridization closed as GenBank accession No. NM_004797, and as (Northern blotting, dot blotting, DNA chip analysis, and for the translation product thereof, amino acid sequence the like), a method of using PCR (RT-PCR, competitive

28 55 EP 3 050 573 A1 56

PCR,real-time PCR, and the like), and thelike. The quan- several-fold volume of a solvent such as a mixture of titative PCR method such as competitive PCR and real- chloroform and methanol to the sample, the Bligh-Dyer time PCR is preferred, since it enables quick and con- method in which a lipid is extracted by adding several- venient detection of change of expression of a gene from fold volume of a solvent such as a mixture of chloroform, an extremely small amount of sample with good quanti- 5 methanol and water, or the like. Further, as for the method fication ability. for detecting the separated lipid, it can be detected by [0156] As for the details of these measurement meth- using a known method such as liquid chromatography ods, they can be performed with reference to, for exam- (LC), gas chromatography (GC), and high performance ple, Molecular Cloning, 2nd edition (J. Sambrook et al., liquid chromatography (HPLC). Alternatively, a method Cold Spring Harbor Lab. Press, 1989). 10 of directly detecting a lipid contained in a fat cell or tissue [0157] Expression of the PPARγ gene in a cell can be may also be used. The reagents and the like usable for investigated by preparing a protein fraction from the cell, such a method are marketed, and for example, HCS Li- and detecting a translation product of the gene (namely, pidTOX Phospholipidosis and Steatosis Detection Kit (In- PPARγ protein) contained in the fraction. PPARγ can be vitrogen) and the like can be used. detected by an immunoassay (for example, ELISA, FIA, 15 [0161] Although change of value of an intracellular in- RIA, Western blotting, and the like) using antibodies that dex measured in the screening method of the present specifically recognize the protein, or it can be detected invention may be increase in the value of intracellular by measuring the activity of the protein using a known index, or may be decrease in the value of intracellular method. Alternatively, the protein can also be detected index, increase in the value of intracellular index is pre- by using mass spectrometry such as MALDI-TOFMS. 20 ferred. [0158] For the details of these general technical means, review articles, published books, and the like can Examples be referred to. For example, "Radioimmunoassay", Ed- ited by H. Irie (Kodansha, 1974), "Radioimmunoassay, [0162] Hereafter, the present invention will be ex- Second Series", Edited by H. Irie (Kodansha, 1979), "En- 25 plained with reference to examples and reference exam- zyme Immunoassay", Edited by E. Ishikawa et al. (Igaku- ples. However, the present invention is not limited by the Shoin, 1978), "Enzyme Immunoassay", 2nd Edition, Ed- examples. ited by E. Ishikawa et al. (Igaku-Shoin, 1982), "Enzyme Immunoassay", 3rd Edition, Edited by E. Ishikawa et al. Example 1: Analysis of suppression of cell proliferation (Igaku-Shoin, 1987), "Methods in ENZYMOLOGY", Vol. 30 and apoptosis of cultured cells of human giant cell tumor 70 (Immunochemical Techniques (Part A)), ibid., Vol. 73 of bone (GCT) observed after culture with addition of zal- (Immunochemical Techniques (Part B)), ibid., Vol. 74 toprofen (Immunochemical Techniques (Part C)), ibid., Vol. 84 (Immunochemical Techniques (Part D: Selected Immu- [0163] As for GCTB cultured cells (case 1, patient with noassays)), ibid., Vol. 92 (Immunochemical Techniques 35 giant cell tumor of bone in a distal part of the right femur, (Part E: Monoclonal Antibodies and General Immu- in twenties; case 2, patient with giant cell tumor of bone noassay Methods)), ibid., Vol. 121 (Immunochemical in a distal part of the right femur, in twenties), cells of Techniques (Part I: Hybridoma Technology and Mono- tumor tissues derived from the patients were cultured by clonal Antibodies)) (these are published by Academic using the DMEM medium containing 2 mM L-glutamine, Press), and the like can be referred to. 40 10% fetal bovine serum (FBS), 100 U/mL of penicillin, [0159] The PPARγ-agonistic activity can be measured and 100 mg/mL of streptomycin with reference to the re- in the same manner as that for the measurement of the port of Cheng YY et al. (Cheng YY, Huang L, Lee KM, PPARγ expression-inducing activity mentioned above, et al., 2004, Bisphosphonates induce apoptosis of stro- i.e., by preparing an RNA fraction, a protein fraction, or mal tumor cells in giant cell tumor of bone, Calcif. Tissue a lipid fraction from a cell, and detecting a transcription 45 Int., 75:71-77), repeatedly subcultured until there were product, or a translation product of a gene locating down- only spindle-shaped cells, which state was gradually at- stream of the PPARγ gene (for example, apoptosis-re- tained from the state that there were also multinucleated lated gene, fat cell differentiation-related gene, arterio- giant cells observed in an early stage, and then used for sclerosis-related gene, anti-inflammation-related gene, the following analysis. As for the analysis of suppression and the like) or a lipid contained in the fraction. The meth- 50 of cell proliferation, the cells were cultured overnight until ods for preparing the RNA fraction and the protein fraction the cells became sub-confluent on a 96-well culture plate and the methods for detecting them may be the same as using the DMEM medium containing 2 mM L-glutamine, the aforementioned methods explained for the measure- 10% fetal bovine serum (FBS), 100 U/mL of penicillin, ment of the PPARγ expression-inducing activity. and 100 m/mL of streptomycin in an incubator of 5% 55 [0160] As for the preparation method of a lipid, it may CO2/95% air at 37°C, zaltoprofen was added to the cells be prepared by using a known method, and there can be at various concentrations (5, 10, 50, 100, and 200 mM), used, for example, the Folch method in which a lipid is color development was attained with Cell Counting Kit- extracted from a sample containing the lipid by adding 8 (CCK-8, Dojindo) 24 hours thereafter, and absorbance

29 57 EP 3 050 573 A1 58 was measured at 450 nm further 3 hours thereafter (Fig. Example 3: Analysis of fat cell differentiation of cultured 1).As a result,zaltoprofen concentration-dependent sup- cells of human giant cell tumor of bone (GCTB) observed pression of the cell proliferation could be confirmed. Zal- after culture with addition of zaltoprofen to the GCTB cul- toprofen was added as a solution in DMSO (dimethyl tured cells sulfoxide) prepared at a concentration 1000 times higher 5 than the final concentration, and the solution was added [0168] It has been reported that PPARγ is a transcrip- in a volume of 0.1% of the volume of the medium. The tion factor indispensable for fat cell differentiation. There- same shall apply to the other drugs used in the following fore, the GCTB cultured cells of the aforementioned case examples. 1 were cultured on chamber cover slide glass, and when [0164] Further, the aforementioned GCTB cultured10 they reached confluent, zaltoprofen was added to the cells of the case 1 mentioned above were cultured on cells at various concentrations (10, 50, and 100m M). chamber cover slide glass, and zaltoprofen was added From 24 hours thereafter, the cells were cultured in a fat at different concentrations (100, and 200 mM) 24 hours cell differentiation-inducing medium (STREMPRO Adi- afterward. Then, 8 hours and 24 hours afterward, the pogenesis Differentiation Kit, Invitrogen) for 7 to 14 days, cells were fixed with 4% paraformaldehyde, staining with 15 and differentiation into fat cells was analyzed with HCS caspase 3 and Tunel assay were performed, and pres- Lipid TOX Green Neutral Lipid Stain (Invitrogen) (Figs. ence or absence of apoptosis was analyzed. In the Tunel 8 and 9). As a result, it was successfully confirmed that assay, fragmented DNAs produced in the process of ap- only a few positive images were obtained with HCS Lipid optosis were detected by the TdT-mediated dUTP nick TOX Green Neutral Lipid Stain for the control, but positive end labeling method (TUNEL). Observation was per-20 images obtained with HCS Lipid TOX Green Neutral Lipid formed with a fluorescence microscope (BZ-9000) of Stain were increased in a zaltoprofen concentration-de- Keyence, and positive images for each concentration pendent manner. were quantitatively observed (Figs. 2 to 5). As a result, [0169] Therefore, it was verified that zaltoprofen ena- zaltoprofen concentration and administration time-de- bles prophylactic treatment or therapeutic treatment of pendent increases of the Tunel-positive ratio and cas- 25 giant cell tumor of bone (GCTB) by differentiating giant pase 3-positive ratio could be confirmed. cell tumor of bone (GCTB) into fat cells, and thereby mak- [0165] Therefore, it was verified that zaltoprofen is use- ing the tumor disappear. ful for prophylactic treatment or therapeutic treatment of giant cell tumor of bone (GCT). It was also verified that Example 4: Analysis of apoptosis of cells derived from suppression of proliferation of giant cell tumor of bone 30 diseased part of patient with giant cell tumor of bone (GCT) by zaltoprofen is based on cell death caused by (GCTB) administered with zaltoprofen apoptosis. [0170] An operational excision sample of a man in his Example 2: PPARγ immunostaining of cultured cells of 30’s who had been administered with 3 tablets per day human giant cell tumor of bone (GCTB) performed after 35 of the zaltoprofen tablets, Soleton Tablet 80 (generic culture with addition of zaltoprofen name: zaltoprofen, 80 mg, Nippon Chemiphar) for about 28 days for pain due to the tumor, and then subjected to [0166] The GCTB cultured cells of the aforementioned the operation, and an operational excision sample of gi- case 1 were cultured on chamber cover slide glass, and ant cell tumor of bone extracted from a patient with giant 24 hours thereafter, zaltoprofen was added at various 40 cell tumor of bone not administered with zaltoprofen concentrations (10, 50, 100, and 200 mM). After 24 hours, (case in which tumor was excised by usual surgical op- the cells were fixed with 4% paraformaldehyde, and im- eration according to standard therapy) as a control were munohistochemical staining of PPARγ was performed. subjected to caspase 3 staining and Tunel assay, and The PPARγ protein was fluorescently detected by using analyzed for the presence or absence of apoptosis. The the antibody SC-7273 of Santa Cruz Biotechnology, Inc. 45 caspase 3 protein was fluorescently detected by using as the primary antibody and the rat anti-mouse IgG FITC Anti-ACTIVE Caspase-3-Ab (Roche) (G7481) (11-4011-85) of eBioscience as the secondary antibody. (12-4739-81) as the primary antibody, and Donkey F2 Observation was performed with a fluorescence micro- Fragment anti-Rabbit IgG PE of eBioscience as the sec- scope (BZ-9000) of Keyence, and positive images for ondary antibody. Observation was performed with a flu- each concentration were quantitatively observed (Figs. 50 orescence microscope (BZ-9000) produced by Keyence 6 and 7). As a result, it was successfully confirmed that (Fig. 10). As a result, almost no Tunel-positive cells and the expression of PPAR γ was increased in a zaltoprofen caspase 3-positive cells could be confirmed among the concentration-dependent manner, while the expression cells derived from the GCTB patient not administered of PPARγ was about 15% in the control. with zaltoprofen, whilst Tunel-positive cells and caspase [0167] Therefore, it was verified that zaltoprofen ena- 55 3-positive cells could be confirmed among the cells de- bles prophylactic treatment or therapeutic treatment of rived from the patient administered with zaltoprofen. giant cell tumor of bone (GCTB) on the basis of promotion [0171] Therefore, it was verified in human that sup- of expression of PPARγ. pression of proliferation of giant cell tumor of bone

30 59 EP 3 050 573 A1 60

(GCTB) by zaltoprofen is based on cell death caused by Example 7: Cell proliferation-inhibitory PPAR effect of apoptosis. zaltoprofen and troglitazone on cultured cells of human giant cell tumor of bone (GCTB) cultured in the presence Example 5: PPAR γ immunostaining of cells derived from or absence of PPARγ siRNA diseased part of patient with giant cell tumor of bone 5 (GCTB) administered with zaltoprofen [0176] The cells (case 1, case 2) were cultured on a 96-well culture plate, allowed to react with PPAR γ siRNA [0172] An operational excision sample of a man in his (Dharmacon, catalog number M-003436-02-0005) that 30’s who had been administered with 3 tablets per day selectively inhibits expression of PPARγ, control siRNA of the zaltoprofen tablets, Soleton Tablet 80 (generic10 (Dharmacon,catalog number D-001206-14-05,final con- name: zaltoprofen, 80 mg, Nippon Chemiphar) for about centration 100 nM), negative control siRNA designed so 28 days for pain due to the tumor, and then subjected to as not to have the gene expression inhibitory action, or the operation, and an operational excision sample of gi- only the transfection reagents (Thermo Scientific Dhar- ant cell tumor of bone excised from a patient with giant maFECT, Thermo Scientific) for 48 hours, and further cell tumor of bone not administered with zaltoprofen15 cultured in a usual culture medium for 48 hours. Then, (case in which tumor was excised by usual surgical op- 200 mMof zaltoprofenor 60 mMof troglitazone was added eration according to standard therapy) as a control were to the cells, 72 hours thereafter, color development was subjected tostaining of PPAR γ, and expression of PPAR γ performed with Cell Counting Kit-8 (CCK-8, Dojindo), and was analyzed by PCR. For PCR, Hs_PPARG_1 SG- 3 hours thereafter, absorbance was measured at 450 nm QuantiTect Primer Assay (200) (QT00029841) of QIA- 20 (Figs. 14 and 15). As a result, it was successfully con- GEN was used. Observation was performed with a fluo- firmed that the cell proliferation-inhibiting effect of zalto- rescence microscope (BZ-9000) of Keyence (Fig. 11). profen and troglitazone was significantly suppressed in As a result, almost no PPARγ-expressing cells could be the PPARγ siRNA addition group. confirmed among the cells derived from the GCTB patient [0177] Therefore, it was verified that zaltoprofen and not administered with zaltoprofen, whilst among the cells 25 troglitazone are useful for prophylactic treatment or ther- derived from the patient administered with zaltoprofen, apeutic treatment of giant cell tumor of bone (GCTB). PPARγ-expressing cells could be confirmed, and in ad- [0178] It was also verified that expression of PPAR γ is dition, fat cell differentiation could be confirmed. indispensable for suppression of proliferation of giant cell [0173] Therefore, it was verified in human that zalto- tumor of bone (GCTB) by zaltoprofen and troglitazone. profen enables prophylactic treatment or therapeutic30 treatment of giant cell tumor of bone (GCTB) by differ- Example 8: Analysis of suppression of cell proliferation entiating giant cell tumor of bone (GCTB) into fat cells on and apoptosis of cells of human giant cell tumor of tendon the basis of promotion of expression of PPAR γ and there- sheath (GCTT) or cells of human pigmented villonodular by making the tumor disappear. synovitis (PVNS) observed after culture with addition of 35 non-steroidal anti-inflammatory agent or thiazolidine de- Example 6: Analysis of suppression of cell proliferation rivative ofcultured cells of human giant cell tumor of bone (GCTB) observed after culture with addition of non-steroidal anti- [0179] In the same manner as that of Example 1, cul- inflammatory agent tured cells of giant cell tumor of tendon sheath (GCTT) 40 (derived from a patient with giant cell tumor of tendon [0174] In the same manner as that of Example 1, the sheath in the right knee, in 30’s, to whom only surgical GCTB cultured cells (case 1, case 2) were cultured, a operation was performed according to standard therapy) non-steroidal anti-inflammatory agent (acetaminophen, and cultured cells of pigmented villonodular synovitis indomethacin, or diclofenac) or troglitazone was added (PVNS) (derived from the patient of case k with pigment- at various concentrations, and absorbance was meas- 45 ed villonodular synovitis in the left knee, in 30’s) were ured at 450 nm (Figs. 12 and 13). As a result, it was cultured from tumor tissues derived from the patients by successfully confirmed that the proliferation of cells was using the DMEM medium containing 2 mM L-glutamine, suppressed in a drug concentration-dependent manner. 10% fetal bovine serum (FBS), 100 U/mL of penicillin, [0175] Therefore, it was verified that these non-steroi- and 100 mg/mL of streptomycin with reference to the re- dal anti-inflammatory agents (acetaminophen, 50 in- port of Cheng YY et al. (Cheng YY, Huang L, Lee KM, domethacin, and diclofenac) and troglitazone enable pro- et al., 2004, Bisphosphonates induce apoptosis of stro- phylactic treatment or therapeutic treatment of giant cell mal tumor cells in giant cell tumor of bone, Calcif Tissue tumor of bone (GCTB). Int., 75:71-77), repeatedly subcultured until there were only spindle-shaped cells, which state was gradually at- 55 tained from the state that there were also multinucleated giant cells observed in an early stage, and then used for the following analysis. As for the analysis of suppression of cell proliferation, the cells were cultured overnight until

31 61 EP 3 050 573 A1 62 they became sub-confluent on a 96-well culture plate us- Example 10: Analysis of fat cell differentiation of cells of ing the DMEM medium containing 2 mM L-glutamine, giant cell tumor of tendon sheath (GCTT) or cells of pig- 10% fetal bovine serum (FBS), 100 U/mL of penicillin, mented villonodular synovitis (PVNS) observed after cul- and 100 mg/mL of streptomycin in an incubator of 5% ture with addition of non-steroidal anti-inflammatory 5 CO2/95% air at 37°C, zaltoprofen (50, 100, 200, 400, and agent 800 mM) or troglitazone (12.5, 25, 50, 100, and 200 mM) wasadded tothe cells,color development was performed [0185] It has been reported that PPARγ is a transcrip- with Cell Counting Kit-8 (CCK-8, Dojindo) 24 hours there- tion factor indispensable for fat cell differentiation. There- after, and absorbance was measured at 450 nm further fore, in the same manner as that of Example 3, zaltopro- 3 hours thereafter (Figs. 16 and 17). As a result, it was 10 fen was added at a concentration of 400 mM to the cells successfully confirmed that the cell proliferation was sup- of giant cell tumor of tendon sheath (GCTT) or cells of pressed in a zaltoprofen or troglitazone concentration- pigmented villonodular synovitis (PVNS), and differenti- dependent manner. ation into fat cells was analyzed (Figs. 30 and 31). As a [0180] Therefore, it was verified that zaltoprofen and result, it was successfully confirmed that the positive im- troglitazone are useful for prophylactic treatment or ther- 15 ages obtained with HCS LipidTOX Green increased in apeutic treatment of giant cell tumor of tendon sheath the zaltoprofen-added cells compared with the control. (GCTT) and pigmented villonodular synovitis (PVNS). [0186] Therefore, it was verified that zaltoprofen ena- [0181] Further, in the same manner as that of Example bles prophylactic treatment or therapeutic treatment of 1, the cultured cells of human giant cell tumor of tendon giant cell tumor of tendon sheath (GCTT) and pigmented sheath (GCTT) and the cultured cells of human pigment- 20 villonodular synovitis (PVNS) by differentiating cells of ed villonodular synovitis (PVNS) were subjected to stain- giant cell tumor of tendon sheath (GCTT) and pigmented ing with caspase 3 and Tunel assay, and presence or villonodular synovitis (PVNS) into fat cells, and thereby absence of apoptosis was analyzed (Figs. 18 to 25). As making the tumor disappear. a result, it was successfully confirmed that the Tunel- positive ratio and caspase 3-positive ratio increased in 25 Example 11: Analysis of suppression of cell proliferation the cells added with zaltoprofen at a concentration of 400 of cultured cells of human giant cell tumor of bone mM or troglitazone at a concentration of 200m M com- (GCTB), cultured cells of giant cell tumor of tendon pared with those observed for the control. sheath (GCTT), and cultured cells of pigmented villon- [0182] Therefore, it was verified that zaltoprofen and odular synovitis (PVNS) observed after culture with ad- troglitazone suppress proliferation of cells of giant cell 30 dition of pioglitazone tumor of tendon sheath (GCTT) and pigmented villonod- ular synovitis (PVNS) on the basis of cell death caused [0187] In the same manner as that of Example 1, the by apoptosis. GCTB cultured cells (case 1, case 2), the cells of giant cell tumor of tendon sheath (GCTT), or the cells of pig- Example 9: PPARγ immunostaining of cells of giant cell 35 mented villonodular synovitis (PVNS) were cultured, and tumor of tendon sheath (GCTT) or cells of pigmented then pioglitazone, which is a thiazolidine derivative, was villonodular synovitis (PVNS) performed after culture added at various concentrations, and absorbance was with addition of non-steroidal anti-inflammatory agent or measured at 450 nm (Fig. 32). As a result, it was suc- thiazolidine derivative cessfully confirmed that the proliferation of cells was sup- 40 pressed in a pioglitazone concentration-dependent man- [0183] In the same manner as that of Example 2, zal- ner. toprofen or troglitazone was added at concentration of [0188] Therefore, it was verified that pioglitazone is 400 mM or 200 mM, respectively, to the cells of giant cell useful for prophylactic treatment or therapeutic treatment tumor of tendon sheath (GCTT) or cells of pigmented of giant cell tumor of bone (GCTB), giant cell tumor of villonodular synovitis (PVNS), and PPAR γ-positive imag- 45 tendon sheath (GCTT), and pigmented villonodular syn- es was confirmed (Figs. 26 to 29). As a result, expression ovitis (PVNS). of PPARγ was successfully observed in the zaltoprofen or troglitazone-added cells. Example 12: Analysis of MRI images of patient with giant [0184] Therefore, it was verified that zaltoprofen and cell tumor of bone (GCTB), patient with giant cell tumor troglitazone enable prophylactic treatment or therapeutic 50 of tendon sheath (GCTT), or patient with pigmented vil- treatment of giant cell tumor of tendon sheath (GCTT) lonodular synovitis (PVNS) administered with zaltopro- and pigmented villonodular synovitis (PVNS) on the basis fen of promotion of expression of PPARγ. [0189] Soleton Tablet 80 (generic name: zaltoprofen, 55 80 mg, Nippon Chemiphar) was administered to a patient with giant cell tumor of bone (GCTB), patient with giant cell tumor of tendon sheath (GCTT), or patient with pig- mented villonodular synovitis (PVNS) at a dose of 3 tab-

32 63 EP 3 050 573 A1 64 lets per day (one tablet was administered in the morning, Example 14: PPARγ immunostaining of cells of chond- at noon, and in the evening), and the tumor size was rosarcoma cell line (H-EMC-SS) performed after culture evaluated by MRI every several months. As a typical with addition of non-steroidal anti-inflammatory agent or case, in a case of recurrence of giant cell tumor of bone thiazolidine derivative (GCTB) in the pelvic part (34 years old, female, Figs. 33 5 and 34), gradual shrinkage of the tumor could be con- [0195] In the same manner as that of Example 2, to firmed after two months (Fig. 35) and four months (Fig. cells of the chondrosarcoma cell line was added zalto- 36). Further, in a case of recurrence of pigmented villon- profen at a concentration of 200m M, troglitazone at a odular synovitis (PVNS) in the right knee (26 years old, concentration of 100 mM, or pioglitazone at a concentra- female, Figs. 37 and 38), attenuation of MRI imaging ef- 10 tion of 200 mM, and the PPARγ-positive images were fect was successfully observed after three months (Figs. observed (Figs. 48 and 49). As a result, expression of 39 and 40), and improvement was observed for pain and PPARγ could be confirmed in the zaltoprofen, troglita- knee-joint excursion. Furthermore, in another case of re- zone, or pioglitazone-added cells. currence of pigmented villonodular synovitis (PVNS) in [0196] Therefore, it was verified that zaltoprofen, tro- the right knee (38 years old, female, Figs. 41 and 42), 15 glitazone, and pioglitazone enable prophylactic treat- shrinkage of tumor could be confirmed, and improvement ment or therapeutic treatment of chondrosarcoma on the of pain could be observed after two months (Figs. 43 and basis of promotion of expression of PPARγ. 44). [0190] Therefore, it was verified in human that zalto- Example 15: Analysis of cell proliferation-suppressing ef- profen is useful for prophylactic treatment or therapeutic 20 fect of various thiazolidinedione derivatives and various treatment of giant cell tumor of bone (GCTB), giant cell non-steroidalanti-inflammatory agents for cells of human tumor of tendon sheath (GCTT), and pigmented villon- giant cell tumor of bone (GCTB), cells of human giant odular synovitis (PVNS). cell tumor of tendon sheath (GCTT), cells of human pig- mentedvillonodular synovitis (PVNS), and cells of human Example 13: Analysis of suppression of cell proliferation 25 chondrosarcoma cell lines and apoptosis of cells of human chondrosarcoma-de- rived cell line (H-EMC-SS) observed after culture with [0197] In the same manners as those of Examples 1, addition of non-steroidal anti-inflammatory agent or thi- 6, 8, 11, and 13, cell proliferation-suppressing effects of azolidine derivative various kinds of thiazolidinedione derivatives and non- 30 steroidal anti-inflammatory agents were analyzed. The [0191] In the same manner as that of Example 1, cells analysis was performed in the same manners as those of the chondrosarcoma cell line were cultured, troglita- of Examples 1 and 6 for cells of giant cell tumor of bone zone, pioglitazone, or zaltoprofen was added at various (GCT-1, GCT-2), in the same manner as that of Example concentrations, and absorbance was measured (Fig. 8 for cells of giant cell tumor of tendon sheath and cells 45). The H-EMC-SS cells were obtained from the Riken 35 of pigmented villonodular synovitis, and in the same man- BioResource Center. As a result, it was successfully con- ner as that of Example 13 for cells of chondrosarcoma firmed that the cell proliferation was suppressed in a tro- cell lines (H-EMC-SS, SW1353), and absorbance was glitazone, pioglitazone, or zaltoprofen concentration-de- measuredat 450 nm. The H-EMC-SScells wereobtained pendent manner. from the Riken BioResource Center. As the SW1353 [0192] Therefore, it was verified that troglitazone, pi- 40 cells, those obtained from ATCC were used. As a result, oglitazone, and zaltoprofen are useful for prophylactic the following results were obtained. treatment or therapeutic treatment of chondrosarcoma. [0198] It was successfully confirmed that pioglitazone [0193] Further, in the same manner as that of Example (50 mm, 100 mm, and 200 mm) suppressed proliferation 1, cells of the chondrosarcoma cell line were subjected of cells of giant cell tumor of bone (GCT-1, GCT-2), cells to caspase 3 staining, and presence or absence of ap- 45 of giant cell tumor of tendon sheath (GCTT), cells of pig- optosis was analyzed (Figs. 46 and 47). As a result, it mented villonodular synovitis, and cells of chondrosar- was successfully confirmed that the caspase 3-positive coma cell lines (H-EMC-SS, SW1353) in a concentration- ratio increased in the cells added with zaltoprofen at a dependent manner (Fig. 50). concentration of 200 mM, troglitazone at a concentration [0199] It was successfully confirmed that troglitazone of 100 mM, or pioglitazone at a concentration of 200 mM, 50 (12.5 mm, 25 mm, 50 mm, 100 mm, and 200 mm) sup- compared with the control. pressed proliferation of cells of giant cell tumor of bone [0194] Therefore, it was verified that suppression of (GCT-1, GCT-2), cells of giant cell tumor of tendon proliferation of chondrosarcoma by troglitazone, piogli- sheath, cells of pigmented villonodular synovitis, and tazone, and zaltoprofen is based on cell death caused cells of chondrosarcoma cell lines (H-EMC-SS, SW1353) by apoptosis. 55 in a concentration-dependent manner (Fig. 51). [0200] It was successfully confirmed that rosiglitazone (50 mm, 100 mm, 200 mm, 400 mm, and 800 mm) sup- pressed proliferation of cells of giant cell tumor of bone

33 65 EP 3 050 573 A1 66

(GCT-1, GCT-2),cells of giantcell tumor oftendon sheath villonodular synovitis, and cells of chondrosarcoma cell (GCTT), cells of pigmented villonodular synovitis, and lines (H-EMC-SS, SW1353) in a concentration-depend- cellsof chondrosarcoma celllines (H-EMC-SS,SW1353) ent manner (Fig. 58). in a concentration-dependent manner (Fig. 52). [0207] It was successfully confirmed that mofezolac [0201] It was successfully confirmed that zaltoprofen 5 (12.5 mm, 25 mm, 50 mm, 100 mm, and 200 mm) sup- (5 mm, 10 mm, 50 mm, 100 mm, and 200 mm for cells of pressed proliferation of cells of giant cell tumor of bone giant cell tumor of bone, 50 mm, 100 mm, 200 mm, 400 (GCT-1, GCT-2), cells of giant cell tumor of tendon sheath mm, and 800 mm for cells of giant cell tumor of tendon (GCTT), cells of pigmented villonodular synovitis, and sheath, cells of pigmented villonodular synovitis, and cells of chondrosarcoma cell lines (H-EMC-SS, SW1353) cells of chondrosarcoma cell lines) suppressed prolifer- 10 in a concentration-dependent manner (Fig. 59). ation of cells of giant cell tumor of bone (GCT-1, GCT- [0208] It was successfully confirmed that acemetacine 2), cells of giant cell tumor of tendon sheath (GCTT), cells (50 mm, 100 mm, 200 mm, 400 mm, and 800 mm) sup- of pigmented villonodular synovitis, and cells of chond- pressed proliferation of cells of giant cell tumor of bone rosarcoma cell lines (H-EMC-SS, SW1353) in a concen- (GCT-1, GCT-2), cells of giant cell tumor of tendon sheath tration-dependent manner (Fig. 53). 15 (GCTT), cells of pigmented villonodular synovitis, and [0202] It was successfully confirmed that diclofenac (5 cells of chondrosarcoma cell lines (H-EMC-SS, SW1353) mm, 10 mm, 50 mm, 100 mm, and 200 mm for cells of giant in a concentration-dependent manner (Fig. 60). cell tumor of bone, 25 mm, 50 mm, and 100 mm for cells [0209] It was successfully confirmed that oxaprozin (50 of giant cell tumor of tendon sheath (GCTT), 25 mm, 50 mm, 100 mm, 200 mm, 400 mm, and 800 mm for cells of mm, 100 mm, 200 mm, and 400 mm for cells of pigmented 20 giant cell tumor of bone, cells of pigmented villonodular villonodular synovitis, and cells of chondrosarcoma cell synovitis, and cells of chondrosarcoma cell lines; 50 mm, lines) suppressed proliferation of cells of giant cell tumor 100 mm, and 200 mm for cells of giant cell tumor of tendon of bone (GCT-1, GCT-2), cells of giant cell tumor of ten- sheath) suppressed proliferation of cells of giant cell tu- don sheath (GCTT), cells of pigmented villonodular syn- mor of bone (GCT-1, GCT-2), cells of giant cell tumor of ovitis, and cells of chondrosarcoma cell lines (H-EMC- 25 tendon sheath (GCTT), cells of pigmented villonodular SS, SW1353)in a concentration-dependent manner (Fig. synovitis, and cells of chondrosarcoma cell lines (H- 54). EMC-SS, SW1353) in a concentration-dependent man- [0203] Itwas successfully confirmed that indomethacin ner (Fig. 61). As for the cells of pigmented villonodular (5 mm, 10 mm, 50 mm, 100 mm, and 200 mm for cells of synovitis, only slight suppression of the proliferation was giant cell tumor of bone; 12.5 mm, 25 mm, and 50 mm for 30 observed at the concentration of 200 mm, and this was cells of giant cell tumor of tendon sheath (GCTT); 12.5 because the number of usable cells was limited, and the mm, 25 mm, 50 mm, 100 mm, and 200 mm for cells of suppression of cell proliferation could not be measured pigmented villonodular synovitis; 50 mm, 100 mm, 200 with such high concentrations as 400 mm or higher. How- mm, 400 mm, and 800 mm for cells of chondrosarcoma ever, on the basis of the effects on the cells of giant cell cell lines) suppressed proliferation of cells of giant cell 35 tumor, cells of pigmented villonodular synovitis, and cells tumor of bone (GCT-1, GCT-2), cells of giant cell tumor of chondrosarcoma cell lines, it was estimated that oxa- of tendon sheath (GCTT), cells of pigmented villonodular prozin also suppresses proliferation of cells of giant cell synovitis, and cells of chondrosarcoma cell lines (H- tumor of tendon sheath (GCTT) at a high concentration EMC-SS, SW1353) in a concentration-dependent man- of 400 mm or higher. ner (Fig. 55). 40 [0210] It was successfully confirmed that acetami- [0204] It was successfully confirmed that celecoxib nophen (50 mm, 100 mm, 200 mm, 400 mm, and 800 mm) (12.5 mm, 25 mm, 50 mm, 100 mm, and 200 mm) sup- suppressed proliferation of cells of giant cell tumor of pressed proliferation of cells of giant cell tumor of bone bone (GCT-1, GCT-2), cells of giant cell tumor of tendon (GCT-1, GCT-2),cells of giantcell tumor oftendon sheath sheath (GCTT), cells of pigmented villonodular synovitis, (GCTT), cells of pigmented villonodular synovitis, and 45 and cells of chondrosarcoma cell lines (H-EMC-SS, cellsof chondrosarcoma celllines (H-EMC-SS,SW1353) SW1353) in a concentration-dependent manner (Fig. in a concentration-dependent manner (Fig. 56). 62). [0205] It was successfully confirmed that etodolac (50 [0211] It was successfully confirmed that lornoxicam mm, 100 mm, and 200 mm) suppressed proliferation of (5 mm, 10 mm, 20 mm, 40 mm, and 80 mm) suppressed cells of giant cell tumor of bone (GCT-1, GCT-2), cells of 50 proliferation of cells of giant cell tumor of bone (GCT-1, giant cell tumor of tendon sheath (GCTT), cells of pig- GCT-2), cells of giant cell tumor of tendon sheath mented villonodular synovitis, and cells of chondrosar- (GCTT), cells of pigmented villonodular synovitis, and coma cell lines (H-EMC-SS, SW1353) in a concentration- cells of chondrosarcoma cell lines (H-EMC-SS, SW1353) dependent manner (Fig. 57). in a concentration-dependent manner (Fig. 63). The de- [0206] It was successfully confirmed that meloxicam 55 gree of the suppression of cell proliferation apparently (50 mm, 100 mm, and 200 mm) suppressed proliferation seems to be low, but this was because the measurement of cells of giant cell tumor of bone (GCT-1, GCT-2), cells could be performed for a concentration of only up to 80 of giant cell tumor of tendon sheath, cells of pigmented mm, which is 1/10 of the drug concentration of acetami-

34 67 EP 3 050 573 A1 68 nophen, due to the low solubility of lornoxicam in DMSO drugs used in the following examples. (dimethyl sulfoxide). [0212] It was successfully confirmed that ampiroxicam Example 17: Analysis of suppression of proliferation of (1.25 mm, 2.5 mm, 5 mm, 10 mm, and 20 mm) suppressed cells of human chondrosarcoma cell line (OUMS-27) cul- proliferation of cells of giant cell tumor of bone (GCT-1, 5 tured in the presence or absence of GW9662 before- GCT-2), cells of giant cell tumor of tendon sheath hand, observed after further culture with addition of zal- (GCTT), cells of pigmented villonodular synovitis, and toprofen, rosiglitazone, or troglitazone cellsof chondrosarcoma celllines (H-EMC-SS,SW1353) in a concentration-dependent manner (Fig. 64). The de- [0218] Cells of the human chondrosarcoma cell line, gree of the suppression of cell proliferation apparently 10 OUMS-27, were cultured in the same manner as that of seems to be low, but this was because the measurement Example 1 until they became sub-confluent, then could be performed for a concentration of only up to 20 GW9662 (Sigma Aldrich, M6191), which is an irreversible mm at the highest due to the low solubility of the drug in antagonist of PPARγ, was added to the cells at a final DMSO (dimethyl sulfoxide). concentration of 1 mM, and the cells were cultured for 60 [0213] It was successfully confirmed that naproxen (50 15 minutes. Zaltoprofen or troglitazone was added to the mm, 100 mm, 200 mm, 400 mm, and 800 mm) suppressed cells at various concentrations of 50, 100, 200, and 400 proliferation of cells of giant cell tumor of bone (GCT-1, mM, or rosiglitazonewas addedat various concentrations GCT-2), cells of giant cell tumor of tendon sheath of 12.5, 25, 50, and 100 mM, color development was per- (GCTT), cells of pigmented villonodular synovitis, and formed with Cell Counting Kit-8 (CCK-8, Dojindo) 72 cellsof chondrosarcoma celllines (H-EMC-SS,SW1353) 20 hours thereafter, absorbance was measured at 450 nm in a concentration-dependent manner (Fig. 65). further 3 hours thereafter, and the result was compared [0214] Therefore, it was verified that pioglitazone, tro- with that observed with adding only DMSO not dissolving glitazone, rosiglitazone, zaltoprofen, diclofenac, in- GW9662 (Fig. 67). As a result, it was successfully con- domethacin, celecoxib, etodolac, meloxicam, mofezolac, firmed that proliferation of cells of human chondrosarco- acemetacin, oxaprozin, acetaminophen, lornoxicam,25 ma cell (OUMS-27) was suppressed by zaltoprofen, tro- ampiroxicam, and naproxen are useful for prophylactic glitazone, or rosiglitazone, and this cell proliferation-sup- treatment or therapeutic treatment of giant cell tumor of pressing action was reduced by GW9662, which is an bone (GCTB), giant cell tumor of tendon sheath (GCTT), irreversible antagonist of PPARγ. pigmented villonodular synovitis (PVNS), and chondro- [0219] Therefore, it was verified that zaltoprofen, tro- sarcoma. 30 glitazone, and rosiglitazone are useful for prophylactic treatment or therapeutic treatment of chondrosarcoma. Example 16: Analysis of suppression of proliferation of It was also verified that zaltoprofen, troglitazone, and ros- cells of human chondrosarcoma (OUMS-27) observed iglitazone suppress proliferation of chondrosarcoma after culture with addition of zaltoprofen, rosiglitazone, through activation of PPARγ. or troglitazone 35 Example 18: Analysis of suppression of proliferation of [0215] Cells of the human chondrosarcoma cell line, cells of human chondrosarcoma cell line (SW1353) cul- OUMS-27 (purchased from ICRB Cell Bank), were cul- tured in the presence or absence of GW9662 before- tured in the same manner as that of Example 1 until they hand, observed after further culture with addition of zal- became sub-confluent, zaltoprofen, rosiglitazone, or tro- 40 toprofen glitazone was added to the cells at a concentration of 50, 100, 200, or 400 mM, color development was performed [0220] Cells of the human chondrosarcoma cell line, with Cell Counting Kit-8 (CCK-8, Dojindo) 72 hours there- SW1353, were cultured in the same manner as that of after, and absorbance was measured at 450 nm further Example 1 until they became sub-confluent, then, 3 hours thereafter as an index of cell count (Fig. 66). As 45 GW9662, which is an irreversible antagonist of PPARγ, a result, it was verified that zaltoprofen and troglitazone was added to the cells at a final concentration of 1 mM, suppressed proliferation of cells of human chondrosar- and the cells were cultured for 60 minutes. Then, zalto- coma (OUMS-27) at concentrations of 200 mM and 400 profen was added to the cells at various concentrations mM, and rosiglitazone suppressed at a concentration of of 100, 200, 300, and 400 mM, color development was 50 mM or higher. 50 performed with Cell Counting Kit-8 (CCK-8, Dojindo) 72 [0216] Therefore, it was verified that zaltoprofen, ros- hours thereafter, absorbance was measured at 450 nm iglitazone, and troglitazone are useful for prophylactic further 3 hours thereafter, and the result was compared treatment or therapeutic treatment of chondrosarcoma. with the absorbance observed with adding only DMSO [0217] As in Example 1, each drug was added as a not dissolving GW9662 (Fig. 68). As a result, it was suc- solution in DMSO (dimethyl sulfoxide) prepared at a con- 55 cessfully confirmed that proliferation of cells of human centration 1000 times higher than the final concentration, chondrosarcoma (SW1353) was suppressed by zalto- and the solution was added in a volume of 0.1% of the profen in a concentration-dependent manner, and the volume of the medium. The same shall apply to the other cell proliferation-suppressing action of zaltoprofen for hu-

35 69 EP 3 050 573 A1 70 man chondrosarcoma cells (SW1353) was markedly re- 003436-02-0005) that selectively suppresses expres- duced by GW9662, which is an irreversible antagonist of sion of PPARγ, or a control siRNA (Dharmacon, catalog PPARγ. number D-001206-14-05) was added to the cells at a [0221] Therefore, it was verified that zaltoprofen is use- final concentration of 100 nM, and the cells were cultured ful for prophylactic treatment or therapeutic treatment of 5 for 48 hours. Then, 400 mM zaltoprofen was added to chondrosarcoma. It was also verified that zaltoprofen the cells, color development was performed with Cell suppresses proliferation of chondrosarcoma through ac- Counting Kit-8 (CCK-8, Dojindo) 24 hours thereafter, ab- tivation of PPARγ. sorbance was measured at 450 nm further 3 hours there- after, and the absorbance observed with the pretreat- Example 19: Analysis of suppression of proliferation of 10 ment with PPARγ siRNA was compared with the absorb- cells of human chondrosarcoma cell line (SW1353) cul- ance observed with the pretreatment with the control siR- tured beforehand in the presence or absence of siRNA NA (Fig. 70). As a result, it was successfully confirmed that suppresses expression of PPARγ, observed after that zaltoprofen suppressed proliferation of cells of hu- further culturewith addition ofzaltoprofen orrosiglitazone man chondrosarcoma (H-EMC-SS), and the cell prolif- 15 eration-suppressing action of zaltoprofen for the human [0222] Cells of the human chondrosarcoma cell line, chondrosarcoma cell line (H-EMC-SS) was completely SW1353, was cultured in the same manner as that of eliminated by siRNA that selectively suppresses expres- Example 1 until they became sub-confluent, PPAR γ-siR- sion of PPARγ. NA (Dharmacon, catalog number M-003436-02-0005) [0225] Therefore, it was verified that zaltoprofen is use- that selectively suppresses expression of PPARγ, or a 20 ful for prophylactic treatment or therapeutic treatment of control siRNA (Dharmacon, catalog number D-chondrosarcoma. It was also verified that expression of 001206-14-05) was added to the cells at a final concen- PPARγ is indispensable for suppression of proliferation tration of 100 nM, and the cells were cultured for 48 hours. of chondrosarcoma by zaltoprofen. Then, 400 mM of zaltoprofen or 100 mM of rosiglitazone was further added to the cells, color development was 25 Example 21: Analysis of suppression of proliferation of performed with Cell Counting Kit-8 (CCK-8, Dojindo) 24 cultured cells of human PVNS cultured beforehand in the hours thereafter, absorbance was measured at 450 nm presence or absence of siRNA that suppresses expres- further 3 hours thereafter, and the absorbance observed sion of PPARγ, observed after further culture with addi- with the pretreatment with PPAR γ siRNA was compared tion of rosiglitazone or zaltoprofen with the absorbance observed with the pretreatment with 30 the control siRNA (double-stranded RNA of non-sense [0226] PVNS cells excised from the patient of Example sequence not having gene expression-suppressing ac- 8 (patient with giant cell tumor of tendon sheath in the tion) (Fig. 69). As a result, it was successfully confirmed right knee, in 30’s, to whom only surgical operation was that 400 mM zaltoprofen suppressed proliferation of cells performed according to standard therapy) were cultured of human chondrosarcoma (SW1353) by about 38%, and 35 in the same manner as that of Example 8 until they be- 100 mM rosiglitazone suppressed proliferation of cells of came sub-confluent, then PPARγ siRNA (Dharmacon, human chondrosarcoma (SW1353) by about 61%. Fur- catalog number M-003436-02-0005) that selectively sup- ther, the cell proliferation-suppressing action of the drugs presses expression of PPAR γ, or a control siRNA (Dhar- forthe human chondrosarcoma cellline was substantially macon, catalog number D-001206-14-05) was added to eliminated by suppressing expression of PPAR γ by using 40 the cells at a final concentration of 100 nM, and the cells siRNA. were cultured for 48 hours. Then, 100 mM of rosiglitazone [0223] Therefore, it was verified that zaltoprofen and or 400 mM of zaltoprofen was added to the cells, color rosiglitazone are useful for prophylactic treatment or ther- development was performed with Cell Counting Kit-8 apeutictreatment of chondrosarcoma. Itwas also verified (CCK-8, Dojindo) 24 hours thereafter, absorbance was that expression of PPARγ is indispensable for suppres- 45 measured at 450 nm further 3 hours thereafter, and the sion of proliferation of chondrosarcoma by zaltoprofen absorbance observed with the pretreatment with PPAR γ and rosiglitazone. siRNA was compared with the absorbance observed with the pretreatment with the control siRNA (Fig. 71). As a Example 20: Analysis of suppression of proliferation of result, it was successfully confirmed that the cell prolif- cells of human chondrosarcoma cell line (H-EMC-SS) 50 eration-suppressing action of rosiglitazone or zaltoprofen culturedbeforehand in the presence or absence of siRNA for the human PVNS cells was substantially completely that suppresses expression of PPARγ, observed after eliminated by siRNA that selectively suppresses expres- further culture with addition of zaltoprofen sion of PPARγ. [0227] Therefore, it was verified that zaltoprofen and [0224] Cells of the human chondrosarcoma cell line, 55 rosiglitazone are useful for prophylactic treatment or ther- H-EMC-SS, were cultured in the same manner as that apeutic treatment of PVNS. It was also verified that ex- of Example 1 until they became sub-confluent, PPARγ pression of PPARγ is indispensable for suppression of siRNA (Dharmacon, catalog number proliferation M- of PVNS by zaltoprofen or rosiglitazone.

36 71 EP 3 050 573 A1 72

Example 22: Analysis of suppression of proliferation of higher than 80%, and 100 mM troglitazone suppressed cells of human bone sarcoma cell line (HOS) observed the cell migration of cells of the human chondrosarcoma after culture with addition of zaltoprofen cell line (SW1353) by about 65%. Further, 400 mM zal- toprofen also suppressed the cell migration of cells of the [0228] Cells of the human bone sarcoma cell line, HOS 5 human chondrosarcoma cell line (SW1353) by about (purchased from American Type Culture Collection), 40%. were cultured in the same manner as that of Example 1 [0231] Therefore, it was verified that acetaminophen, until they became sub-confluent. Zaltoprofen was added celecoxib, indomethacin, diclofenac, rosiglitazone, trogl- to the cells at concentrations of 25, 50, 100, 200 and 400 itazone, zaltoprofen, and pioglitazone are useful for pre- mM, color development was performed with Cell Count- 10 vention of metastasis of chondrosarcoma. ing Kit-8 (CCK-8, Dojindo) 72 hours thereafter, and ab- sorbance was measured at 450 nm further 3 hours there- Example 24: Analysis of cell invasion of cells of human after (Fig. 72). As a result, it was successfully confirmed chondrosarcoma cell line (SW1353) observed after cul- that the cell proliferation of the cells of the human bone ture with addition of non-steroidal anti-inflammatory sarcoma cell line (HOS) was suppressed by zaltoprofen 15 agent or PPARγ agonist at concentrations of 100 mM, 200 mM, and 400 mM in a concentration-dependent manner. In particular, zaltopro- [0232] For the analysis of cell invasion, Matrigel™ In- fen could suppress proliferation of the cells of the human vasion Chamber (BD Bioscience, catalog number bone sarcoma cell line (HOS) by about 60% at the con- 354480), which uses Matrigel as the matrix, was used. centration of 400 mM. 20 Cells of the human chondrosarcoma cell line, SW1353, [0229] Therefore, it was verified that zaltoprofen is use- were cultured in the same manner as that of Example 1 ful for prophylactic treatment or therapeutic treatment of until they became sub-confluent, and then each of trogl- bone sarcoma. itazone (50 mM and 100 mM), pioglitazone (200 mM), zal- toprofen (200 mM, 300 mM, and 400 mM), diclofenac (200 Example 23: Analysis of cell migration of cells of human 25 mM), rosiglitazone (100 mM and 200 mM), acetami- chondrosarcoma cell line (SW1353) observed after cul- nophen (200 mM), indomethacin (200 mM),and celecoxib ture with addition of non-steroidal anti-inflammatory (50 mM) was added to the cells at each of the final con- agent or PPARγ agonist centrations mentioned in the parentheses, the cells were fixed with 4% paraformaldehyde 24 hours afterward, and [0230] The analysis of cell migration was performed 30 stained with hematoxylin, and number of invaded cells by using a 6-well plate of TPP according to the method was counted. As a result, it was successfully confirmed described in Takeuchi A, et al., Cancer Science,that all the compounds suppressed the cell invasion of 104:740-749, 2013 (Low molecular weight heparin sup- cells of the human chondrosarcoma cell line (SW1353) presses receptor for advanced glycation end products- atall the set concentrations in a concentration-dependent mediated expression of malignant phenotype in human 35 manner (Fig. 75). In particular, 100 mM troglitazone and fibrosarcoma cells). Cells of the human chondrosarcoma 50 mM celecoxib completely suppressed the cell invasion cell line, SW1353, were cultured in the same manner as of cells of the human chondrosarcoma cell line that of Example 1 until they became sub-confluent, and (SW1353), and 400 mM zaltoprofen also suppressed the the cells were delaminated in a width of 1 mm using a cell invasion to lower than 20%. Figs. 76 and 77 show micropipette tip (200 ml). Each of acetaminophen (200 40 microphotographs of invaded cells fixed by immersion mM and 400 mM), celecoxib (25 mM, 50 mM, and 75 mM), into a 4% paraformaldehyde solution, and stained with indomethacin (100 mM and 200 mM), diclofenac (100 mM hematoxylin (magnification is 200 times). From the mi- and 200 mM), rosiglitazone (200 mM and 400 mM), trogl- crophotographs of Figs. 76 and 77, the patterns of the itazone (50 mM and 100 mM), zaltoprofen (100 mM, 200 invaded cells can be understood. With the conditions of mM, and 400 mM), and pioglitazone (100 mM and 200 45 addition of 100 mM troglitazone or 50 mM celecoxib, there mM) was added to the cells at each of the final concen- were cells in such a number that they only sparsely ex- trations mentioned in the parentheses, and the cells were isted, and they rolled into small balls. Therefore, it can fixed with 4% paraformaldehyde 72 hours afterward, and be seen that not only the suppression of cell invasion, stained with Crystal Violet. Then, area of the cells migrat- but also cell injury was realized. ed to the region where the cells were delaminated was 50 [0233] Therefore, it was verified that troglitazone, pi- measured by using Image J software (http://rsb.in- oglitazone, zaltoprofen, diclofenac, rosiglitazone, aceta- fo.nih.gov/ij/index.html) (Figs. 73 and 74). As a result, it minophen, indomethacin, and celecoxib are useful for was successfully confirmed that all the compounds sup- prevention of metastasis of chondrosarcoma. pressed the cell migration of cells of the human chond- rosarcoma cell line (SW1353) at all the set concentra- 55 tions in a concentration-dependent manner. In particular, 75 mM celecoxib suppressed the cell migration of cells of the human chondrosarcoma cell line (SW1353) by

37 73 EP 3 050 573 A1 74

Example 25: Analysis of cell invasion of cells of human cell tumor of bone including 8 male patients from 21 years chondrosarcomacell line (SW1353) cultured beforehand old to 39 years old and 5 female patients from 25 years in the presence or absence of GW9662 observed after old to 68 years old. As for the affected parts, 4 patients further culture with addition of zaltoprofen affected at the pelvic part, 2 patients at distal part of thigh- 5 bone, 1 patient at proximal part of fibula (2 patients, if 1 [0234] Cells of the human chondrosarcoma cell line, patient affected as the primary lesion of metastasis to SW1353, were cultured in the same manner as that of the lung is included), 1 patient at distal part of tibia, 1 Example 1 until they became sub-confluent, then, patient at knee part, 1 patient at proximal part of humerus, GW9662, which is an irreversible antagonist of PPARγ, 1 patient at sacrum, and 2 patients with metastases to was added to the cells at a final concentration of 1 mM, 10 the lung (1 patient affected at proximal part of fibula, and and the cells were cultured for 60 minutes. Then, zalto- 1 patient affected at proximal part of tibia as primary le- profen was added to the cells at various concentrations sions). The patients affected at the pelvic part were all of 200, 300, and 400 mM, cell invasion was measured female patients, and the patients affected at the distal with Matrigel™ Invasion Chamber (BD Bioscience, cat- part of thighbone or proximal part of fibula were all male alog number 354480) 24 hours afterward, and the result 15 patients. There were 5 patients with recurrence. In par- was compared with the degree of cell invasion observed ticular, the tumor of the patient of case f (25 years old, with addition of only DMSO not dissolving GW9662 (Fig. female, proximal part of right humerus) was of the 5th 78). As a result, it was successfully confirmed that the recurrence, and the tumor of the patient of the case a (34 cell invasion of human chondrosarcoma cells (SW1353) years old, female, pelvis) was of the 3rd recurrence. Fur- was suppressed by higher than 80% with 400 mM zalto- 20 ther, there were two patients having metastases to the profen, and the suppression of the cell invasion of the lung (cases e and m), and in particular, the patient of the human chondrosarcoma cells (SW1353) with 400m M case m (21 years old, male, proximal part of right tibia) zaltoprofen was substantially eliminated by GW9662 (1 suffered from multiple metastases to the lung. mM), which is an irreversible antagonist of PPARγ. [0238] For the patient of the case j (32 years old, male, [0235] Therefore, it was verified that zaltoprofen is use- 25 sacrum), administration of Zometa (registered trade- ful for prevention of metastasis of chondrosarcoma. Fur- mark), which is zoledronic acid hydrate injection, and ar- ther, it was also verified that zaltoprofen prevents metas- tery embolization were used in combination. For the pa- tasis of chondrosarcoma through activation of PPARγ. tient of the case m (21 years old, male, proximal part of right tibia, multiple metastases to lung), Zometa (regis- Example 26: Analysis of curative effect of zaltoprofen 30 tered trademark), which is zoledronic acid hydrate injec- (Soleton Tablet (registered trademark) on patient with tion, was used in combination, after treatment with de- giant cell tumor of bone nosumab, which is an anti-RANKL human monoclonal antibody. For the patient of the case b (32 years old, [0236] Soleton Tablet 80 (generic name: zaltoprofen, female, pelvis), a treatment with zaltoprofen was per- 80 mg, Nippon Chemiphar) was administered to patients 35 formed after completion of the clinical trial with denosum- with giant cell tumor of bone at a dose of 3 tablets per ab. day (one tablet was administered in the morning, at noon, [0239] As a result of the administration of zaltoprofen, and in the evening). Four weeks after the start of the as shown in Figs. 79 and 80, among the 13 cases of the administration, the tumor size was evaluated by imaging patients of giant cell tumor of bone, they were diagnosed based on simple Roentgen contrast method, X-ray CT, 40 partial response (PR) in one case, stable disease (SD) and MRI (nuclear magnetic resonance imaging). Then, in 11 cases, and progressive disease (PD) in one case. the tumors were excised from patients for whom surgical In the case a, for which diagnosis was partial response operation was possible, administration of zaltoprofen (PR), the tumor size shrank by more than 70%, the giant was continued at a dose of 240 mg per day (one 80 mg celltumor of bone was then excised by surgical operation, tablet was administered at the time of breakfast, lunch, 45 and no recurrence has been observed up to today. The and supper), and the tumor size was evaluated by imag- patient of the case m (21 years old, male, proximal part ing based on simple Roentgen contrast method, X-ray of right tibia), evaluated as progressive disease (PD), CT, and MRI (nuclear magnetic resonance imaging) eve- suffered from multiple metastases to the lung, and was ry 16 weeks. Also for patients for whom surgical operation temporarily evaluated as stable disease (SD), but the was difficult, administration of zaltoprofen was continued 50 tumor tends to increase again with progress of time. As at a dose of 240 mg per day (one 80 mg tablet was ad- for the patient of the case 1 (31 years old, male, left knee ministered at the time of breakfast, lunch, and supper), part), who showed 19.8% of increase of the size of the and the tumor size was evaluated by imaging based on giant cell tumor of bone, it has been found that he took simple Roentgen contrast method, X-ray CT, and MRI only about a half of the prescribed dose according to the (nuclear magnetic resonance imaging) every 8 weeks. 55 judgment of the patient himself. [0237] The cases for which therapeutic treatment with [0240] For all the patients of the cases a, c, d, e, and zaltoprofen was performed are summarized in Fig. 79. f, whose giant cell tumors of bone shrank, any treatment Zaltoprofen was administered to total 13 patients of giant was not performed except for the administration of zal-

38 75 EP 3 050 573 A1 76 toprofen, and therefore it was considered that the tumors [0248] Unexpectedly, improving tendency of the KPS were shrank as a result of the taking of zaltoprofen. score was also observed in patients who showed slightly [0241] Transversal MRI images of the affected part growing tendency of giant cell tumor of bone. In the pa- (proximal part of left fibula) of the patient of the case c tient of the case k, in spite of the growth of the giant cell obtained before the administration of zaltoprofen (De- 5 tumor of bone by about 10%, the KPS score improved cember 13, 2012) and after the administration (February from a state that "considerable clinical symptoms, but 13, 2013) are shown in Fig. 81. It can be seen that, as a normal activities are possible with efforts" (score 80) to result of the taking of zaltoprofen over about nine weeks, a state that "slight clinical symptoms, and normal activi- the diameter of the giant cell tumor of bone shrank from ties are possible" (score 90). Similarly, in the patient of 23.3 mm to 21.0 mm. 10 the case j, in spite of the growth of the giant cell tumor [0242] Frontal and transversal MRI images and CT im- of bone by about 6%, the KPS score dramatically im- ages of the affected part (distal part of right tibia) of the proved from a state that "nursing and periodical medical patient of the case d obtained before the administration intervention are needed in consideration of conditions of of zaltoprofen (July 4, 2013) and after the administration disease" (score 50) to a state that "slight clinical symp- (August 29, 2013) are shown in Fig. 82. It can be seen 15 toms, and normal activities are possible" (score 90). that, as a result of the taking of zaltoprofen over about [0249] A further surprising finding was that, even for eight weeks, the diameter of the giant cell tumor of bone the patient of giant cell tumor of bone of the case m (21 shrank from 21.7 mm to 20.5 mm. years old, male, proximal part of right tibia), who suffered [0243] Transversal MRI images of the affected part from multiple metastases to the lung, and evaluated as (metastasis part in the lung) of the patient of the case e 20 progressive disease (PD), the KPS score improved from obtained before the administration of zaltoprofen (Janu- a state that "slight clinical symptoms, and normal activi- ary 17, 2013) and after the administration (September ties are possible" (score 90) to a state of "no clinical symp- 19, 2013) are shown in Fig. 83. It can be seen that, as a toms" (score 100). result of the taking of zaltoprofen over about 35 weeks, [0250] Further, there was no case in which the KPS the diameter of the giant cell tumor of bone metastasized 25 score worsened among the cases of giant cell tumor of to the lung shrank from 8.2 mm to 7.3 mm. bone in which the patients took zaltoprofen. [0244] As described above, shrinkage of giant cell tu- [0251] In addition, since there were 6 cases (cases a, mor of bone could be confirmed in the patients who took b, d, g, i, and j) where observation of hardening of bones zaltoprofen. Further, in the 12 cases except for one case was brought by the taking of zaltoprofen as determined in which multiple metastases to the lung were seen, the 30 on the basis of X-ray CT images, it was estimated that giant cell tumors of bone were evaluated as partial re- taking of zaltoprofen restored the bones, and improved sponse (PR) or stable disease (SD). Further, shrinkage mechanical strength thereof. of giant cell tumor of bone metastasized to the lung was [0252] As described above, administration of zaltopro- also observed (case e, Fig. 83). fen provided shrinkage or arrest of growth of tumors of [0245] Then, because improving tendency was also 35 the giant cell tumor of bone patients, observation of hard- seen for subjective symptoms of the patients, ability to ening of bones, and marked improvement in ability of the perform everyday activities was also evaluated accord- patients to carry out everyday activities. Further, any new ing to the Karnofsky Performance Status (KPS). KPS is metastasis of giant cell tumor of bone was not observed an evaluation method for classifying patient’s conditions during the taking of zaltoprofen. Furthermore, no recur- into ten stages of score 100 to 0 according to the criteria 40 rence of giant cell tumor of bone could be confirmed dur- shown in Fig. 84, and a higher score means better per- ing the taking of zaltoprofen in the patients who were formance of the patient for everyday activities. The eval- subjected to an ablative operation of giant cell tumor of uation results of KPS of the patients of giant cell tumor bone. of bone before and after the administration of zaltoprofen [0253] Therefore, it was verified that zaltoprofen is use- are shown in Fig. 85. 45 ful for prophylactic treatment, therapeutic treatment, and [0246] In the patients of the cases a, c, and d, whose prevention of metastasis of giant cell tumor of bone in giant cell tumors of bone shrank as a result of the taking human patients. Further, it was also verified that zalto- of zaltoprofen, the KPS scores were improved from a profen restores or forms bones of human patients suffer- state that "considerable clinical symptoms, but normal ing from giant cell tumor of bone, and improves ability to activities are possible with efforts" (score 80) to a state 50 carry out everyday activities. that "slight clinical symptoms, and normal activities are possible" (score 90). Example 27: Analysis of curative effect of zaltoprofen [0247] In the patient of the case g, the giant cell tumor (Soleton Tablet (registered trademark)) on PVNS pa- of bone did not shrink, but it also did not grow, and the tients KPS scores were markedly improved from a state that 55 "considerableclinical symptoms,but normalactivities are [0254] PVNS patients were administered with 3 tablets possible with efforts" (score 80) to a state of "no clinical per day (one tablet was administered in the morning, at symptoms" (score 100). noon, and in the evening) of Soleton Tablet 80 (generic

39 77 EP 3 050 573 A1 78 name; zaltoprofen, 80 mg, Nippon Chemiphar). Four [0259] Sagittal MRI images of the affected part (right weeks after the start of the administration, the tumor size knee joint) of the patient of the case c obtained before was evaluated by imaging based on simple Roentgen theadministration of zaltoprofen (December6, 2012) and contrast method, X-ray CT, and MRI (nuclear magnetic after the administration (August 1, 2013) are shown in resonance imaging). Then, the tumors were excised from 5 Fig. 89. It can be seen that, as a result of the taking of patients for whom surgical operation was possible, ad- zaltoprofen over about 35 weeks, the diameter of PVNS ministration of zaltoprofen was continued at a dose of shrank from 59.5 mm to 48.0 mm. 240 mg per day (one 80 mg tablet was administered at [0260] As described above, shrinkage of PVNS could the time of breakfast, lunch, and supper), and the tumor be confirmed in all the patients who took zaltoprofen. size was evaluated by imaging based on simple Roent- 10 Further, all the PVNS patients who took zaltoprofen were gen contrast method, X-ray CT, and MRI (nuclear mag- judgedto be partialresponse (PR) or stable disease (SD). netic resonance imaging) every 16 weeks. Also for pa- [0261] The evaluation results of the PVNS patients ob- tients for whom surgical operation was difficult, adminis- tained before and after the administration of zaltoprofen tration of 240 mg zaltoprofen per day (one 80 mg tablet according to the Karnofsky Performance Status (KPS) was administered at the time of breakfast, lunch, and 15 are shown in the table of Fig. 90. In the patients of the supper)was continued, andthe tumor size was evaluated cases a and b whose PVNS shrank as a result of the by imaging based on simple Roentgen contrast method, taking of zaltoprofen, the KPS score was dramatically X-ray CT, and MRI (nuclear magnetic resonance imag- improved from a state that "patient can care himself or ing) every 8 weeks. herself, but normal activities or works are impossible" [0255] The cases for which therapeutic treatment with 20 (score 70) to a state of "no clinical symptoms" (score zaltoprofen was performed are summarized in Fig. 86. 100). Similarly, in the patients of the cases c and d whose Zaltoprofen was administered to total 14 PVNS patients PVNS shrank as a result of the taking of zaltoprofen, the including 5 male patients from 26 years old to 65 years KPS score was improved from a state that "considerable old and 9 female patients from 16 years old to 62 years clinical symptoms, but normal activities are possible with old. As for the affected parts, 8 patients affected at the 25 efforts" (score 80) to a state that "slight clinical symptoms, knee joint, 4 patients at ankle joint, 1 patient at shoulder and normal activities are possible" (score 90). joint, and 1 patient at wrist joint. The case of 1 patient [0262] Further, there was no case in which the KPS who affected PVNS at the wrist joint was that of focal score worsened among the cases of PVNS in which the PVNS, and all the other 13 cases were diffuse PVNS patients took zaltoprofen. cases. Ten cases out of the 14 cases were those of re- 30 [0263] As described above, as a result of the taking of currence of PVNS. As shown in Figs. 86 and 87, among zaltoprofen, tumors of the PVNS patients shrank, or 11 cases for which the response rate was successfully growth of the tumors was arrested, and ability to carry determined, one case was judged to be partial response out everyday activities was markedly improved. Further, (PR), and the other 10 cases were judged to be stable during the taking of zaltoprofen, any new metastasis of disease (SD). 35 PVNS was not observed. Furthermore, no recurrence of [0256] The patient of the case h (16 years old, female, PVNScould be confirmed duringthe taking of zaltoprofen right ankle joint), the patient of the case i (26 years old, in the patients who were subjected to an ablative oper- male, right knee), and the patient of the case e (40 years ation of PVNS. old, male, left wrist joint) were subjected to PVNS ablative [0264] Therefore, it was verified that zaltoprofen is use- operation. Further, for the patient of the case 1 (31 years 40 ful for prophylactic treatment, therapeutic treatment, and old, female, left ankle joint), the patient of the case m (30 prevention ofmetastasis ofPVNS inhuman patients. Fur- years old, female, left knee), and the patient of the case ther, it was also verified that zaltoprofen improves ability n (38 years old, female, ankle joint), the administration to carry out everyday activities of human patients who of zaltoprofen was started after the PVNS ablative oper- suffer from PVNS. ation, and therefore the response rate was not success- 45 [0265] On the basis of the examples mentioned above, fully determined. it was verified that the pharmaceutical agent of the [0257] All the patients of the cases a, b, c, d, e, and f present invention containing a substance having a whose PVNS shrank were not subjected to any treatment PPARγ-agonistic activity and/or a PPAR γ expression-in- except for the administration of zaltoprofen, and accord- ducing activity as an active ingredient is useful as an ingly it was considered that the tumors were shrunk by 50 agent for prophylactic treatment, therapeutic treatment, the administration of zaltoprofen. or prevention of metastasis of giant cell tumor occurring [0258] Frontal MRI images of the affected part (shoul- in bone and soft tissue, chondrosarcoma, or bone sar- der joint) of the patient of the case a obtained before the coma. administration of zaltoprofen (September 6, 2012) and after the administration (August 20, 2013) are shown in 55 Industrial Applicability Fig. 88. It can be seen that, as a result of the taking of zaltoprofen over about 50 weeks, the diameter of PVNS [0266] The agent for prophylactic treatment, therapeu- shrank from 27.7 mm to 7.9 mm. tic treatment, or prevention of metastasis of the present

40 79 EP 3 050 573 A1 80 invention is effective for patients of giant cell tumor oc- 5. The agent for prophylactic treatment, therapeutic curring in bone and soft tissue, chondrosarcoma, or bone treatment, or prevention of metastasis according to sarcoma, or persons with a possibility of developing giant any one of claims 1 to 4, wherein the substance hav- cell tumor occurring in bone and soft tissue, chondrosa- ing a PPARγ-agonistic activity and/or a PPARγ ex- rcoma, or bone sarcoma. Further, according to the5 pression-inducing activity consists of one or more present invention, search for a novel therapeutic agent kinds of angiotensin II receptor antagonists having for giant cell tumor occurring in a bone and soft tissue, a PPARγ agonistic activity selected from the group chondrosarcoma, or bone sarcoma is enabled by choos- consisting of irbesartan and telmisartan. ing a test substance that controls a PPARγ gene and apoptosis, or fat cell differentiation. 10 6. The agent for prophylactic treatment, therapeutic treatment, or prevention of metastasis according to any one of claims 1 to 5, wherein the substance hav- Claims ing a PPARγ-agonistic activity and/or a PPARγ ex- pression-inducing activity consists of one or more 1. An agent for prophylactic treatment, therapeutic15 kinds of endogenous PPAR γ agonists selected from treatment, or prevention of metastasis of giant cell the group consisting of 15-deoxy-Δ12,14-prostagla- tumor occurring in a bone and soft tissue, chondro- din J2, 15-hydroxyeicosatetraenoic acid, 9-hydrox- sarcoma, or bone sarcoma, which comprises a sub- yoctadecadienoic acid, 13-hydroxyoctadecadienoic stance having a PPARγ-agonistic activity and/or a acid, nitrolinoleic acid, and a long chain fatty acid. PPARγ expression-inducing activity as an active in- 20 gredient. 7. The agent for prophylactic treatment, therapeutic treatment, or prevention of metastasis according to 2. The agent for prophylactic treatment, therapeutic any one of claims 1 to 6, wherein the giant cell tumor treatment, or prevention of metastasis according to occurring in a bone and soft tissue is selected from claim 1, wherein the substance having a PPAR γ-ag- 25 the group consisting of giant cell tumor of bone, giant onistic activity and/or a PPAR γ expression-inducing cell tumor of tendon sheath, and pigmented villon- activity consists of one or more kinds of PPAR γ ag- odular synovitis. onists selected from the group consisting of a non- steroidal anti-inflammatory agent, a thiazolidinedi- 8. The agent for prophylactic treatment, therapeutic one derivative, an angiotensin II receptor antagonist 30 treatment, or prevention of metastasis according to having a PPARγ-agonistic activity, and an endog- any one of claims 1 to 7, which further contains an enous PPARγ agonist. anti-RANKL antibody.

3. The agent for prophylactic treatment, therapeutic 9. The agent for prophylactic treatment, therapeutic treatment, or prevention of metastasis according to 35 treatment, or prevention of metastasis according to claim 1 or 2, wherein the substance having a PPAR γ- claim 8, wherein the anti-RANKL antibody is deno- agonistic activity and/or a PPAR γ expression-induc- sumab. ing activity consists of one or more kinds of non- steroidal anti-inflammatory agents selected from the 10. The agent for prophylactic treatment, therapeutic group consisting of zaltoprofen, diclofenac, in-40 treatment, or prevention of metastasis according to domethacin, proglumetacin, indometacin farnesil, any one of claims 1 to 9, which further contains a celecoxib, etodolac, meloxicam, mofezolac, bisphosphonate. acemetacin, oxaprozin, acetaminophen, lornoxi- cam, ampiroxicam, piroxicam, naproxen, loxopro- 11. The agent for prophylactic treatment, therapeutic fen, rofecoxib, ethenzamide, diflunisal, aluminopro- 45 treatment, or prevention of metastasis according to fen, nabumetone, ketoprofen, acetylsalicylic acid, claim 10, wherein the bisphosphonate consists of ibuprofen, pranoprofen, and sulindac. one or more kinds of bisphosphonates selected from the group consisting of etidronate, clodronate, tiludr- 4. The agent for prophylactic treatment, therapeutic onate, pamidronate, neridronate, olpadronate, alen- treatment, or prevention of metastasis according to 50 dronate, ibandronate, tiludronate, incadronate, rise- any one of claims 1 to 3, wherein the substance hav- dronate, minodronate, zoledronate, solvadronate, ing a PPARγ-agonistic activity and/or a PPARγ ex- medronate, risendronate, amino-olpadronate, si- pression-inducing activity consists of one or more madronate, pyridronate, rezidronate, EB1053, and kinds of thiazolidinedione derivatives selected from YH 529. the group consisting of troglitazone, rosiglitazone, 55 pioglitazone, balaglitazone, rivoglitazone, isaglita- 12. A local infusion for artery embolization or artificial zone, netoglitazone, lobeglitazone, englitazone, and bone, which comprises a substance having a ciglitazone. PPARγ-agonistic activity and/or a PPARγ expres-

41 81 EP 3 050 573 A1 82

sion-inducing activity as an active ingredient. further contains an anti-RANKL antibody.

13. The local infusion for artery embolization or artificial 19. The local infusion for artery embolization or artificial bone according to claim 12, wherein the substance bone according to claim 18, wherein the anti-RANKL having a PPARγ-agonistic activity and/or a PPARγ 5 antibody is denosumab. expression-inducing activity consists of one or more kinds of PPARγ agonists selected from the group 20. The local infusion for artery embolization or artificial consisting of a non-steroidal anti-inflammatory bone according to any one of claims 12 to 19, which agent, a thiazolidinedione derivative, an angiotensin further contains a bisphosphonate. II receptor antagonist having a PPAR γ-agonistic ac- 10 tivity, and an endogenous PPARγ agonist. 21. The local infusion for artery embolization or artificial bone according to claim 20, wherein the bisphos- 14. The local infusion for artery embolization or artificial phonate consists of one or more kinds of bisphos- bone according to claim 12 or 13, wherein the sub- phonates selected from the group consisting of etid- stance having a PPARγ-agonistic activity and/or a 15 ronate, clodronate, tiludronate, pamidronate, nerid- PPARγ expression-inducing activity consists of one ronate, olpadronate, alendronate, ibandronate, or more kinds of non-steroidal anti-inflammatory tiludronate, incadronate, risedronate, minodronate, agents selected from the group consisting of zalto- zoledronate, solvadronate, medronate, risendro- profen, diclofenac, indomethacin, proglumetacin, in- nate, amino-olpadronate, simadronate, pyridronate, dometacin farnesil, celecoxib, etodolac, meloxicam, 20 rezidronate, EB1053, and YH 529. mofezolac, acemetacin, oxaprozin, acetaminophen, lornoxicam, ampiroxicam, piroxicam, naproxen, 22. A method for screening for an agent for prophylactic loxoprofen, rofecoxib, ethenzamide, diflunisal, alu- treatment, therapeutic treatment, or prevention of minoprofen, nabumetone, ketoprofen, acetylsalicyl- metastasis of giant cell tumor occurring in a bone ic acid, ibuprofen, pranoprofen, and sulindac. 25 and soft tissue, chondrosarcoma, or bone sarcoma, which comprises the following steps: 15. The local infusion for artery embolization or artificial bone according to any one of claims 12 to 14, where- (1) the step of culturing a cell or tissue derived in the substance having a PPARγ-agonistic activity from giant cell tumor occurring in a bone and and/or a PPARγ expression-inducing activity con- 30 soft tissue, chondrosarcoma, or bone sarcoma sists of one or more kinds of thiazolidinedione deriv- in the presence or absence of a test substance, atives selected from the group consisting of troglita- (2) the step of measuring one or more kinds of zone, rosiglitazone, pioglitazone, balaglitazone, indices selected from the group consisting of rivoglitazone, isaglitazone, netoglitazone, lobeglita- those defined in (a) to (g) mentioned below in zone, englitazone, and ciglitazone. 35 the presence or absence of the test substance;

16. The local infusion for artery embolization or artificial (a) one or more indices selected from the bone according to any one of claims 12 to 15, where- group consisting of gene expression in the substance having a PPARγ-agonistic activity amount of PPARγ, and protein amount of and/or a PPARγ expression-inducing activity con- 40 PPARγ, sists of one or more kinds of angiotensin II receptor (b) one or more indices selected from the antagonists having a PPARγ agonistic activity se- group consisting of gene expression lected from the group consisting of irbesartan and amount of an apoptosis-related gene, pro- telmisartan. tein amount of a translation product of an 45 apoptosis-related gene, and biological ac- 17. The local infusion for artery embolization or artificial tivity of a translation product of an apopto- bone according to any one of claims 12 to 16, where- sis-related gene, in the substance having a PPARγ-agonistic activity (c) one or more indices selected from the and/or a PPARγ expression-inducing activity con- group consisting of gene expression sists of one or more kinds of endogenous PPARγ 50 amount of a fat cell differentiation-related agonists selected from the group consisting of 15- gene, protein amount of a translation prod- deoxy-Δ12,14-prostagladin J2, 15-hydroxyeicosa- uct of a fat cell differentiation-related gene, tetraenoic acid, 9-hydroxyoctadecadienoic acid, 13- and biological activity of a translation prod- hydroxyoctadecadienoic acid, nitrolinoleic acid, and uct of a fat cell differentiation-related gene, a long chain fatty acid. 55 (d) one or more indices selected from the group consisting of gene expression 18. The local infusion for artery embolization or artificial amount of an arteriosclerosis-related gene, bone according to any one of claims 12 to 17, which protein amount of a translation product of

42 83 EP 3 050 573 A1 84

an arteriosclerosis-related gene, and bio- logical activity of a translation product of an arteriosclerosis-related gene, (e) one or more indices selected from the group consisting of gene expression5 amount of an anti-inflammation-related gene, protein amount of a translation prod- uct of an anti-inflammation-related gene, and biological activity of a translation prod- uct of an anti-inflammation-related gene, 10 (f) an index consisting of a PPAR γ-agonistic activity that can promote transcription of one or more kinds of genes selected from the group consisting of an apoptosis-related gene, a fat cell differentiation-related gene, 15 an arteriosclerosis-related gene, and an an- ti-inflammation-related gene, (g) amount of lipid contained in a fat cell or fat tissue, 20 and (3) the step of selecting a test substance that changes a value or values of the intracellular index or indices in the presence of a test sub- stance compared with the value or values of the 25 intracellular index or indices observed in the ab- sence of the test substance.

23. The screening method according to claim 22, where- in the giant cell tumor occurring in a bone and soft 30 tissue is selected from the group consisting of giant cell tumor of bone, giant cell tumor of tendon sheath, and pigmented villonodular synovitis.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• JP 2005200419 A [0043] [0058] • JP 2006501136 A [0048] [0058] • JP 2009533467 A [0043] [0058] • WO 2008026729 A [0058] • JP 2005343802 A [0048] [0058]

Non-patent literature cited in the description

• Jpn. J. Cancer Res., 1999, vol. 90, 75-80 [0059] • Pharmaceutics. Nankodo Co., Ltd, 1997 [0105] • Cancer Lett., 2010, vol. 297, 65-74 [0059] • Japan Pharmaceutical Excipient Standards 2003. • FEBS Lett., 1999, vol. 455, 135-139 [0059] Yakuji Nippo, August 2003 [0105] • Zhongguo yishi zazhi, 2010, vol. 12 (6), 743-747 • J. SAMBROOK et al. Molecular Cloning. Cold Spring [0059] Harbor Lab. Press, 1989 [0156] • Mol. Pharmacol., 2007, vol. 72, 674-685 [0059] • Radioimmunoassay. 1974 [0158] • British Journal of Cancer, 2002, vol. 86, 1303-1309 • Radioimmunoassay. 1979 [0158] [0059] • Enzyme Immunoassay. 1978 [0158] • Medical Oncology, 2006, vol. 23 (3), 393-402 [0059] • Enzyme Immunoassay. 1982 [0158] • Biochem. Pharmacol., 2011, vol. 82, 464-475 [0059] • Enzyme Immunoassay. 1987 [0158] • Tumor Biology, 2010, vol. 31, 427-436 [0059] • Methods in ENZYMOLOGY. vol. 70 [0158] • J. Pharmacol. Exp. Ther., 2002, vol. 302, 18-25 • METHODS IN ENZYMOLOGY, vol. 73 [0158] [0059] • METHODS IN ENZYMOLOGY, vol. 74 [0158] • The Journal of the Japanese Orthopedic Association, • METHODS IN ENZYMOLOGY. vol. 84 [0158] 2012, vol. 86 (8), 1319, , 2-9, 18 [0059] • METHODS IN ENZYMOLOGY, vol. 92 [0158] • The Journal of the Japanese Orthopedic Association, • METHODS IN ENZYMOLOGY. Academic Press, vol. 2013, vol. 87 (8), 1-8, 22 [0059] 121 [0158] • The Journal of the Japanese Orthopedic Association, • CHENG YY ; HUANG L ; LEE KM et al. Bisphospho- 2013, vol. 87 (6), 1-2, FP3-8 [0059] nates induce apoptosis of stromal tumor cells in giant • Anticancer Research, 2013, vol. 33, 2169-2174 cell tumor of bone. Calcif. Tissue Int., 2004, vol. 75, [0059] 71-77 [0163] • Journal of the Japanese Orthopedic Association, • CHENG YY ; HUANG L ; LEE KM et al. Bisphospho- 2013, vol. 87 (8), 1-8, 20 [0059] nates induce apoptosis of stromal tumor cells in giant • Handbook of Pharmaceutical Additives. Maruzen cell tumor of bone. Calcif Tissue Int., 2004, vol. 75, Co., Ltd, 1989 [0105] 71-77 [0179] • Encyclopedia of Pharmaceutical Additive 2007. • TAKEUCHI A et al. Cancer Science, 2013, vol. 104, Yakuji Nippo, July 2007 [0105] 740-749 [0230]

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