US 2010O216747A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0216747 A1 Ochiai et al. (43) Pub. Date: Aug. 26, 2010

(54) PREVENTIVE AGENT OR THERAPEUTIC Related U.S. Application Data AGENT FOR DISEASE CAUSED BY (60) Provisional application No. 60/935,262, filed on Aug. ABNORMAL BONE METABOLISM 2, 2007. (75) Inventors: Eiji Ochiai, Hino-shi (JP); Miyuki Publication Classification Nishiga, Hino-shi (JP); Kenichiro (51) Int. Cl. Takagi, Hino-shi (JP); Yoshiaki A6II 3/663 (2006.01) Azuma, Hino-shi (JP) A63/675 (2006.01) A6IP 9/08 (2006.01) Correspondence Address: (52) U.S. Cl...... 514/89: 514/108: 514/107: 514/94. SUGHRUE MION, PLLC 2100 PENNSYLVANIA AVENUE, N.W., SUITE (57) ABSTRACT 8OO An object of the present invention is to provide a preventive WASHINGTON, DC 20037 (US) drug and a therapeutic drug for diseases caused by abnormal bone metabolism, especially , which is more (73) Assignee: TEIJIN PHARMA LIMITED, effective than conventional drugs. Combined use of N-hy Chiyoda-ku, Tokyo (JP) droxy-4-5-4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phe noxylpentoxy-benzamidine, or a salt thereof; and at least (21) Appl. No.: 12/671,414 one compound selected from the group consisting of , , , tiludronic (22) PCT Filed: Aug. 1, 2008 acid, , , , , and salts thereof can exert higher bone (86). PCT No.: PCT/UP2008/063864 resorption inhibitory effect and provide a preventive effect and a therapeutic effect for diseases caused by abnormal bone S371 (c)(1), metabolism, especially osteoporosis as compared with (2), (4) Date: Apr. 26, 2010 administration of the respective agents respectively. Patent Application Publication Aug. 26, 2010 Sheet 1 of 3 US 2010/0216747 A1

Figure 1

Effect of combined use of Compound A (10M) and s risedronic acid (3 x 10M) s (5 4.O 8 20 s 9 100 80 2 60

t 40 g 2O

added and concentration PBS Risedronic acid 3 x 10M Comparative Comparative Comparative Example 1-1 Example 1-2 Example 1-3 Example 1 Patent Application Publication Aug. 26, 2010 Sheet 2 of 3 US 2010/0216747 A1

Figure 2)

GSS Effect of combined use of Compound A (10M) and 9. s risedronic acid (3 x 10M) g O

NS. O O S. -- 3.d 8n s d bO

GD c) H d R DMSO Compound A 10M DMSO Compound A O'M Compound added and concentration PBS Risedronic acid 3 x 10'M Comparative Comparative Comparative Example 2-1 Example 2-2 Example 2-3 Example 2 Patent Application Publication Aug. 26, 2010 Sheet 3 of 3 US 2010/0216747 A1

Figure 3

Effect of combined use of Compound A (10M) and clodronic acid (10M) 140 120 OO 80 60 40 20 O Compound DMSO Compound A OM DMSO Compound A 10M added and awa-no-mimesms concentration PBS Clodronic acid Comparative Comparative Comparative 1 OM- 5 Example 3-1 Example 3-2 Example 3-3 Example 3 US 2010/0216747 A1 Aug. 26, 2010

PREVENTIVE AGENT OR THERAPEUTIC 0006 Patent Document 1: Japanese Unexamined Patent AGENT FOR DISEASE CAUSED BY Application Publication No. 2004-537549 ABNORMAL BONE METABOLISM 0007 Non-patent Document 1: Lee Sung-eun, Synthesis and Biological Activity of Natural Products and Designed New Hybrid Compounds for the Treatment of LTB4 Related TECHNICAL FIELD Disease, Graduate School of Pusan National University, 0001. The present invention relates to a preventive or Department of Science, Doctoral dissertation, 1999.8 therapeutic agent for diseases caused by abnormal bone 0008. Non-patent Document 2: R Samad fam et al., Endo metabolism, particularly to a preventive or therapeutic agent crinology, Vol. 148, No. 6, 2007 for osteoporosis, and specifically to a preventive or therapeu 0009. Non-patent Document 3: DM Blacket al., The New tic agent for diseases caused by abnormal bone metabolism, England Journal of Medicine, Vol. 349, No. 13, 2003 especially osteoporosis, containing, as active ingredients, N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl) DISCLOSURE OF THE INVENTION phenoxylpentoxy-benzamidine, or a salt thereof, and at least Problems to be Solved by the Invention one compound selected from the group consisting of 0010. An object of the present invention is to provide a etidronic acid, clodronic acid, pamidronic acid, tiludronic drug or a therapeutic or preventive method that is more effec acid, risedronic acid, minodronic acid, ibandronic acid, tive as a therapeutic or preventive agent and a therapeutic or Zoledronic acid, and salts thereof. preventive method for diseases caused by abnormal bone metabolism, especially osteoporosis than existing drugs. 0011. The present inventors have keenly studied ingredi BACKGROUND ART ents that are effective as a therapeutic or preventive agent for 0002 N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3- diseases caused by abnormal bone metabolism, especially thiazol-4-yl)phenoxylpentoxy-benzamidine represented by osteoporosis, and found that combined use of specific drugs, formula (I) or a salt thereof has a bone resorption inhibitory each of which can be used alone, can provide a therapeutic or effect and a bone formation promoting effect, and is expected preventive agent or a therapeutic or preventive method for diseases caused by abnormal bone metabolism, especially as a therapeutic or preventive agent for osteoporosis. osteoporosis that has extremely high efficacy. 0012. In other words, the present invention is a preventive Formula 1 ortherapeutic agent or a preventive or therapeutic method for diseases caused by abnormal bone metabolism, especially osteoporosis, containing as active ingredients, the following X. N -OH (a) and (b): S ea I0013 (a) N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3- NH2 thiazol-4-yl)phenoxylpentoxy-benzamidine or a salt thereof, and 1n 1a1n 0014 (b) at least one compound selected from the group consisting of etidronic acid, clodronic acid, pamidronic acid, , risedronic acid, minodronic acid, 0003 N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3- ibandronic acid, Zoledronic acid, and salts thereof. thiazol-4-yl)phenoxylpentoxy-benzamidine, or a salt 0015. Further, the present invention is a preventive or thereof is disclosed in Patent Document 1 and Non-patent therapeutic agent for diseases caused by abnormal bone Document 1. metabolism, especially osteoporosis, containing as active 0004 Etidronic acid, clodronic acid, pamidronic acid, ingredients (a) and (b), in a form of a combination drug or tiludronic acid, risedronic acid, minodronic acid, ibandronic independent single drugs. acid, Zoledronic acid, or salts thereof are considered to act specifically on activated and Suppress bone Effect of the Invention resorption by Suppressing their activity, and used as therapeu 0016. The present invention is a preventive or therapeutic tics agent for diseases caused by abnormal metabo drug or a preventive or therapeutic method for diseases lism Such as osteoporosis and the like. caused by abnormal bone metabolism, especially osteoporo 0005. A large number of therapeutic agents for osteoporo sis, wherein N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3- sis are currently available. Therapeutic effects of combined thiazol-4-yl)phenoxylpentoxy-benzamidine, or a salt use of these agents are varied, however. For example, a com thereof, and at least one compound selected from the group bination of PTH (parathyroid hormone) and a certain bispho consisting of etidronic acid, clodronic acid, pamidronic acid, sphonic acid is known to cancel their therapeutic effect each tiludronic acid, risedronic acid, minodronic acid, ibandronic other. Although both agents are representative therapeutic acid, Zoledronic acid, and salts thereof; are administered in agents for osteoporosis, it has been reported as the results of combination, by which an extremely strong preventive or a preclinical study and a clinical study that their effects were therapeutic effect can be obtained as compared with the effect reduced when they were administered in combination as obtained by either ingredient alone. compared with the effects obtained when they were admin istered alone, respectively (Non-patent Documents 2 and 3). BRIEF DESCRIPTION OF DRAWINGS It is not easily conceivable to discover a therapeutic or pre 0017 FIG. 1 is a graph showing an inhibitory effect on the ventive method having higher efficacy using an existing or number of formed -like cells of a solvent, N-hy novel therapeutic agent for osteoporosis, as is also considered droxy-4-5-4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phe from this example. noxylpentoxy-benzamidine (Compound A) and risedronic US 2010/0216747 A1 Aug. 26, 2010

acid that were used respectively or in combination of two of Zoledronic acid, and salts thereof. The combined use provides them in the experimental system in which osteoclast-like cells a stronger effect than the use of the respective ingredients were formed by adding 1C.25(OH), D to mouse bone mar alone as a preventive or therapeutic agent or a preventive or row-derived cells and culturing the cells for 7 days. therapeutic method for osteoporosis. 0018 FIG. 2 is a graph showing the inhibitory effect on the 0025 Among etidronic acid, clodronic acid, pamidronic number of formed osteoclast-like cells of a solvent, N-hy acid, tiludronic acid, risedronic acid, minodronic acid, iban droxy-4-5-4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phe dronic acid, Zoledronic acid, or a salt thereof used in the noxylpentoxy-benzamidine (Compound A) and risedronic present invention, clodronic acid or risedronic acid is prefer acid that were used respectively or in combination of two of able, and risedronic acid is more preferable. When these are them in the experimental system in which osteoclast-like cells used in combination with N-hydroxy-4-5-4-(5-isopropyl were formed by adding 1C.25(OH), D to mouse bone mar 2-methyl-1,3-thiazol-4-yl)phenoxylpentoxy-benzamidine, row-derived cells and culturing the cells for 7 days. ora salt thereof in the present invention, one kind or a mixture 0019 FIG.3 is a graph showing the inhibitory effect on the of two or more kinds in an arbitrary ratio may be used. These number of formed osteoclast-like cells of a solvent, N-hy are manufactured by known methods. droxy-4-5-4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phe 0026. Examples of the salt of etidronic acid, clodronic noxylpentoxy-benzamidine (Compound A) and risedronic acid, pamidronic acid, tiludronic acid, risedronic acid, mino acid that were used respectively or in combination of two of dronic acid, ibandronic acid or Zoledronic acid used in the them in the experimental system in which osteoclast-like cells present invention include pharmaceutically acceptable salts were formed by adding 1C.25(OH)Dto mouse bone mar obtained by using inorganic acids (hydrochloric acid, hydro row-derived cells and culturing the cells for 7 days. bromic acid, Sulfuric acid and phosphoric acid) and organic acids (citric acid, acetic acid, lactic acid, tartaric acid, fumaric BEST MODE FOR CARRYING OUT THE acid, formic acid, propionic acid, oxalic acid, trifluoroacetic INVENTION acid, methanesulfonic acid, benzoic acid, maleic acid, glu conic acid, glycolic acid, Succinic acid, 4-toluenesulfonic 0020 N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3- acid, galacturonic acid, embonic acid, glutamic acid or aspar thiazol-4-yl)phenoxylpentoxy-benzamidine, or a salt tic acid) or inorganic bases (alkaline metal salts of Sodium, thereof used in the present invention has an excellent bone potassium and the like, alkaline-earth metal salts of magne resorption inhibitory effect and a bone formation promoting sium, calcium and the like, and metal salts of aluminum, Zinc effect and is expected as a therapeutic or preventive agent for and the like) or organic bases (such as ammonia, triethy osteoporosis. lamine, diethylamine, ethylenediamine, propanediamine, 0021 N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3- pyrrolidine, piperidine, piperazine, pyridine, lysine, choline, thiazol-4-yl)phenoxylpentoxy-benzamidine may be manu ethanolamine, diethanolamine, N,N-dimethylethanolamine, factured by known methods such as the method described in 4-hydroxypiperidine, glucosamine, N-methylglucamine and Non-patent Document 1 and the like. the like). 0022. Examples of the salt of N-hydroxy-4-5-4-(5-iso 0027. The preventive or therapeutic agent for osteoporosis propyl-2-methyl-1,3-thiazol-4-yl)phenoxylpentoxy-benza according to the present invention provides a drug having an midine include pharmaceutically acceptable salts obtained by effect to improve bone mineral density and bone strength using inorganic acids (hydrochloric acid, hydrobromic acid, equal to or higher than that of conventionally known thera Sulfuric acid and phosphoric acid) and organic acids (citric peutic agents for osteoporosis by using two types of ingredi acid, acetic acid, lactic acid, tartaric acid, fumaric acid, for ents having different mode of actions, namely, at least one mic acid, propionic acid, oxalic acid, trifluoroacetic acid, compound selected from the group consisting of etidronic methanesulfonic acid, ethanesulfonic acid, benzoic acid, acid, clodronic acid, pamidronic acid, tiludronic acid, maleic acid, gluconic acid, glycolic acid, Succinic acid, risedronic acid, minodronic acid, ibandronic acid, Zoledronic 4-toluenesulfonic acid, galacturonic acid, embonic acid, acid, and salts thereof; and N-hydroxy-4-5-4-(5-isopropyl glutamic acid or aspartic acid). In the present invention, 2-methyl-1,3-thiazol-4-yl)phenoxylpentoxy-benzamidine, hydrochloric acid is preferably used as an inorganic acid and or a salt thereof, as active ingredients. The drug according to methanesulfonic acid or ethanesulfonic acid as an organic the present invention is less toxic and has excellent stability. acid. The combined use of the active ingredients allows a reduction 0023 The dose of N-hydroxy-4-5-4-(5-isopropyl-2- in dose as compared with the respective active ingredients methyl-1,3-thiazol-4-yl)phenoxylpentoxy-benzamidine, or used respectively. a salt thereof is an effective dose for the prevention or treat 0028. The dose of at least one compound selected from the ment of osteoporosis and depends on the age and body weight group consisting of etidronic acid, clodronic acid, pamidronic of a patient, the type of combination therapy, the frequency of acid, tiludronic acid, risedronic acid, minodronic acid, iban treatment, the type of desired effect, the administration dronic acid, Zoledronic acid, and salts thereof is an effective method or the like, and thus is not determined uncondition dose for the prevention or treatment of osteoporosis and ally. Generally, when it is used as a therapeutic or preventive depends on the age and body weight of a patient, the type of agent, the ordinary dose administered may be used. combination therapy, the frequency of treatment, the type of 0024. 4-5-4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl) desired effect, the administration method or the like, and thus phenoxylpentoxy-benzamidine, or a salt thereof, which is a is not determined unconditionally. Generally, when it is used dehydroxy form of N-hydroxy-4-5-4-(5-isopropyl-2-me as a therapeutic or preventive agent, the ordinary dose admin thyl-1,3-thiazol-4-yl)phenoxylpentoxy-benzamidine, also istered may be used. has excellent bone resorption inhibitory effect and bone for 0029. The preventive ortherapeutic agent or preventive or mation promoting effect and is expected as a preventive or therapeutic method for osteoporosis according to the present therapeutic agent for osteoporosis. Likewise, 4-5-4-(5-iso invention may be any, as long as it contains N-hydroxy-4-(5- propyl-2-methyl-1,3-thiazol-4-yl)phenoxylpentoxy-benza 4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxypen midine, or a salt thereof is also used in combination with at toxy-benzamidine, or a salt thereof; and at least one com least one compound selected from the group consisting of pound selected from the group consisting of etidronic acid, etidronic acid, clodronic acid, pamidronic acid, tiludronic clodronic acid, pamidronic acid, tiludronic acid, risedronic acid, risedronic acid, minodronic acid, ibandronic acid, acid, minodronic acid, ibandronic acid, Zoledronic acid, and US 2010/0216747 A1 Aug. 26, 2010

salts thereof, as active ingredients. These ingredients may be powder are mixed together at the time of administration. The mixed and simultaneously administered, of may be adminis method for mixing the ingredients immediately before tered separately at the same time or Successively or with a administration may be conducted manually or use a package certain time interval. When not administered simultaneously, that allows mixing the ingredients easily by cutting, drawing, the active ingredients may be administered alternatively, or splitting, pulling out or the like. The forms of the combination one agent may be administered continuously and then another drug include dosage forms such as tablets, pills, granules, agent may be administered, for example. powders, Solutions, Suspensions, syrups, capsules or the like. 0030 The drug according to the present invention may be in any form of a drug, as long as it contains N-hydroxy-4-(5- 0033. Further, the preventive or therapeutic agent for 4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxypen osteoporosis according to the present invention may be toxy-benzamidine, or a salt thereof; and at least one com administered daily or intermittently, and once daily or 2 to 3 pound selected from the group consisting of etidronic acid, times daily with a divided dose. When both active ingredients clodronic acid, pamidronic acid, tiludronic acid, risedronic are single drugs, the number of doses for the respective active acid, minodronic acid, ibandronic acid, Zoledronic acid, and ingredients may be the same or different from each other. salts thereof, as active ingredients. For example, a combina tion drug containing both active ingredients may be consti Examples of the administration method includes a method in tuted, or a single drug containing each of the active ingredi which N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3-thia ents may be constituted. Used herein, "combination drug’ Zol-4-yl)phenoxylpentoxy-benzamidine, or a salt thereof is refers to a preparation containing two or more active ingre administered once daily and at least one compound selected dients in combination in one preparation, and 'single drug from the group consisting of etidronic acid, clodronic acid, refers to a preparation containing one active ingredient in one pamidronic acid, tiludronic acid, risedronic acid, minodronic preparation. According to the aspect of a single drug, when a acid, ibandronic acid, Zoledronic acid, and salts thereof is plurality of compounds selected from etidronic acid, clo administered once weekly or biweekly or twice monthly. dronic acid, pamidronic acid, tiludronic acid, risedronic acid, Further, when both drugs are single drugs, they may be minodronic acid, ibandronic acid and Zoledronic acid or salts administered at ordinary intervals for the respective drugs in thereofare used, the respective compounds may be used as a combination. Even when it is selected to administer the single drug or a combination drug, but it is preferable to use respective drugs at different intervals, convenience is them as single drugs. expected to be improved by using the form of a kit Such as 0031 Atherapeutic or preventive agent or a therapeutic or blister package and the like. preventive method according to the present invention in which both active ingredients are used as single drugs means 0034) N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3- a drug or a method in which single drugs that can be used thiazol-4-yl)phenoxylpentoxy-benzamidine, or a salt respectively are used in combination. Accordingly, drugs thereof, and at least one compound selected from the group containing each of the active ingredients may be in different consisting of etidronic acid, clodronic acid, pamidronic acid, dosage forms. For example, the forms of the respective drugs tiludronic acid, risedronic acid, minodronic acid, ibandronic may be solid or liquid for both, or a combination of solid and acid, Zoledronic acid, and salts thereof according to the liquid, and are not particularly restricted. When the respective present invention may be formulated themselves alone or active ingredients are single drugs, they may be in a form of a together with appropriate excipients described below by kit containing a set of both single drugs. Examples of the known methods. Specific examples of the dosage form representative form of the kit include a blister package in include oral preparations such as Soft capsules, hard capsules, which both drugs in the quantities corresponding to a specific tablets, syrups and the like; parenteral injections; and drugs period (for example, one week or a longer period) in accor for external use. dance with an administration schedule are packaged in one sheet. Further, the drugs may be packed in the same package 0035 Examples of the excipients include vegetable oils like PTP at the end of manufacturing stage of the drugs or may (for example, corn oil, cotton seed oil, coconut oil, almond be placed in the same bag at the time of prescription at a oil, and peanut oil), oily esters such as medium chain fatty hospital or a pharmacy, and the form of the kit is not particu acid glycerides and the like, mineral oils, Vaseline, animal larly limited. fats and oils, cellulose derivatives (for example, crystalline 0032. As the combination drug, for example, both active cellulose, hydroxypropyl cellulose, hydroxypropylmethyl ingredients in the amounts with which the respective ingre cellulose, and methyl cellulose), polyvinyl pyrrolidone, dex dients can exhibit their respective effects may be combined to trin, lactose, mannitol, Sorbitol, starch and the like. In addi manufacture dosage forms such as tablets, capsules, liquids tion, additives Such as antioxidants, wetting agents, viscosity and solution, and the like. The time of combining the active stabilizers, colorants and the like may be added, as required. ingredients to produce a combination drug may be in the stage 0036 Further, the present inventions is not restricted to a of manufacturing a dosage form as a combination drug or preventive or therapeutic agent or a preventive or therapeutic immediately before administration. When the active ingredi method for osteoporosis and may be applied to a preventive or ents are combined in the stage of manufacturing, the active therapeutic agent or a preventive or therapeutic method for ingredients in appropriate amounts may be mixed, shaped, diseases caused by abnormal bone metabolism, especially and packaged. The method of shaping is not particularly diseases caused by abnormal bone resorption among abnor restricted, and the respective agents may be mixed or layered. mal bone metabolism. The diseases caused by abnormal bone When a combination drug is prepared immediately before resorption include rheumatoid arthritis, bone Paget's disease, administration, various methods are available: the respective hypercalcemia, periodontal bone loss, renal osteodystrophy, active ingredients are stored separately until immediately osteolytic tumor, bone metastatic tumor and the like. The before administration, and agents in the liquid form are explanation about the preventive or therapeutic agent or the mixed, or an agent in the Solid form such as a tablet, a pill, preventive ortherapeutic method for osteoporosis can also be granules, powder, or a capsule is dissolved in an agent in the applied to the preventive or therapeutic agent or the preven liquid form, or agents in the Solid form Such as granules or tive or therapeutic method for these diseases. US 2010/0216747 A1 Aug. 26, 2010

Examples the medium was replaced with fresh 10% FCS-OMEM con Example 1 taining the test compound(s) of the respective conditions in both the Comparative Examples and Example. Study of the effects of N-hydroxy-4-5-4-(5-isopro 0039. In both the Comparative Examples and Example, on pyl-2-methyl-1,3-thiazol-4-yl)phenoxylpentoxy the 7th day after the start of culture, the medium was removed, benzamidine and Risedronic Acid on the Formation the cells were fixed with phosphate buffered formalin and of Osteoclast-Like Cells stained by tartrate-resistant acid phosphatase (abbreviated as 0037 Multinuclear osteoclast-like cells were formed from TRAP hereinbelow), an osteoclast marker, using Sigma Acid mouse bone marrow-derived cells by collecting bone mar Phosphatase, Leukocyte (TRAP) kit (Sigma, No. 386A), and rows of the femur and the tibia from a mouse, removing the number of TRAP-positive multinuclear cells having 6 or erythrocytes according to the ordinary method, plating the more cell nuclei was counted as osteoclast-like cells under an residual components in a 96-well plate at the density of optical microscope. 4x10 cells/well, culturing the cells overnight, then adding a medium containing 1C.25-dihydroxy (referred to 0040. The results are shown in FIG. 1. In the graph, the as 1=.25(OH),D, hereinbelow) in order to be at 10M, and percentages of the number of osteoclast-like cells formed in culturing the cells in the presence of 1C,25(OH)D for 7 each of the Comparative Examples and Example, taking the days. An CMEM medium containing 10% fetal calf serum number of osteoclast-like cells formed in Comparative (referred to as 10% FCS-OMEM hereinbelow) was used as a Example 1-1 as 100%, are shown using the values shown as medium. When 1 O.25(OH)D was added to the mouse bone the averagetstandard deviation of 3 wells for each group. In marrow-derived cells, each of a solvent alone (Comparative Comparative Example 1-2 (in which N-hydroxy-4-5-4-(5- Example 1-1), N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1, isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxylpentoxy 3-thiazol-4-yl)phenoxypentoxy-benzamidine (described benzamidine (Compound A) alone was added) and Compara as Compound Ain Figure) alone (Comparative Example 1-2), tive Example 1-3 (in which risedronic acid alone was added), risedronic acid alone (Comparative Example 1-3), and N-hy the numbers of osteoclast-like cells showed a tendency to droxy-4-5-4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phe decrease (Comparative Example 1-2: 81.3% and Compara noxylpentoxy-benzamidine (described as Compound A in tive Example 1-3:88.1%) as compared with that in Compara Figure) and risedronic acid simultaneously (Example 1) were tive Example 1-1 (the control group in which the solvent was added to the mouse bone marrow-derived cell culture system, added), with no significant difference. and the effects on formation of osteoclast-like cells were 0041. As shown in FIG. 1, the number of osteoclast-like evaluated. The following groups were set for the experiment. cells greatly decreased in Example 1 (in which N-hydroxy 4-5-4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxylyl) Comparative Example 1-1 phenoxylpentoxy-benzamidine (Compound A) and risedronic acid were added simultaneously) (52.3%) as com Control Group (Control) pared with those in Comparative Example 1-2 and Compara Comparative Example 1-2 tive Example 1-3, and was significantly lower than that in Comparative Example 1-1. In other words, when the above Group in which N-hydroxy-4-5-4-(5-isopropyl-2- two agents were used in combination, a significantly stronger methyl-1,3-thiazol-4-yl)phenoxylpentoxy-benzami effect was observed than the effect expected from the sum of dine (Compound A) at 107M Alone was Added the effects of the respective single agents. Comparative Example 1-3 0042. In the Figure, * shows p-0.05 as compared with the control group by the Dunnett's t-test: *** shows p-0.001 as Group in which Risedronic Acid at 3x107M Alone compared with the control group by the Dunnett's t-test; it was Added shows p-0.05 as compared with Comparative Example 2-2 by the Student's t-test; and tilt shows p-0.01 as compared Example 1 with Comparative Example 2-3 by the Student's t-test. Group in which N-hydroxy-4-5-4-(5-isopropyl-2-methyl 0043. The osteoclast-like cell formation inhibition rates of 1.3-thiazol-4-yl)phenoxylpentoxy-benzamidine (Com the respective groups with respect to Comparative Example pound A) at 107M and risedronic acid at 3x107 M were 1-1 are shown in Table 1. added simultaneously 0038. For addition of N-hydroxy-4-5-4-(5-isopropyl-2- TABLE 1 methyl-1,3-thiazol-4-yl)phenoxylpentoxy-benzamidine Effects of N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3-thiazol (Compound A), in both the Comparative Examples and 4-yl)phenoxypentoxy-benzamidine and risedronic acid Example, N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3- on the formation of osteoclast-like cells thiazol-4-yl)phenoxylpentoxy-benzamidine (Compound A) Osteoclast-like cell formation was dissolved in dimethylsulfoxide (DMSO) at a concentra inhibition rate (%) tion of 10 M, and the resultant solution was added to the Comparative O.O medium to make 107 Mata 1000-fold dilution. For addition Example 1-1 of risedronic acid, in both the Comparative Examples and Comparative 18.7 Example 1-2 Example, Sodium risedronate hydrate was dissolved in phos Comparative 11.9 phate buffered saline (PBS) at a concentration of 3x10" M, Example 1-3 and the resultant solution was added to the medium to make Example 1 47.7 3x107 M at a 1000-fold dilution. On the third day after addition of the test compound(s) of the respective conditions, US 2010/0216747 A1 Aug. 26, 2010

Example 2 tive Example 2-2 by the Student's t-test; and shows p-0.01 as compared with Comparative Example 2-3 by the Student's Study of the effects of N-hydroxy-4-5-4-(5-isopro t-teSt. pyl-2-methyl-1,3-thiazol-4-yl)phenoxylpentoxy 0046. The osteoclast-like cell formation inhibition rates of benzamidine and Risedronic Acid on the Formation the respective groups with respect to Comparative Example of Osteoclasts 2-1 are shown in Table 2. 0044) The experiment was conducted in a similar manner to that described in Example 1, except that the concentration TABLE 2 of N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3-thiazol-4- Effects of N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3-thiazol yl)phenoxylpentoxy-benzamidine in the medium was set at 4-yl)phenoxypentoxy-benzamidine and risedronic acid 10M, and the following groups were set. on the formation of osteoclast-like cells Comparative Example 2-1 Osteoclast-like cell formation inhibition rate (%) Control Group (Control) Comparative O.O Example 2-1 Comparative Example 2-2 Comparative SO.O Example 2-2 Group in which N-hydroxy-4-5-4-(5-isopropyl-2- Comparative 11.9 methyl-1,3-thiazol-4-yl)phenoxylpentoxy-benzami Example 2-3 dine (Compound A) at 10 MAlone was Added Example 2 89.2 Comparative Example 2-3 Example 3 Group in which Risedronic acid at 3x107M Alone Study of the effects of N-hydroxy-4-5-4-(5-isopro was Added pyl-2-methyl-1,3-thiazol-4-yl)phenoxylpentoxy benzamidine and clodronic acid on the formation of Example 2 osteoclast-like cells Group in which N-hydroxy-4-5-4-(5-isopropyl-2-methyl 0047 The experiment was conducted in a similar manner 1.3-thiazol-4-yl)phenoxylpentoxy-benzamidine (Com to that described in Example 1, except that clodronic acid at a pound A) at 10M and Risedronic Acid at 3x107M were concentration of 10 M was allowed to act on the cells in Added Simultaneously place of risedronic acidata concentration of 3x107M in the 0045. The results are shown in FIG. 2. In the graph, the Comparative Examples and Example, and the following percentages of the number of osteoclast-like cells formed in groups were set. each of the Comparative Examples and Example, taking the number of osteoclast-like cells formed in Comparative Comparative Example 3-1 Example 2-1 as 100%, are shown using the values shown as Control Group (Control) the averagetstandard deviation of 3 wells for each group. In Comparative Example 2-2 (in which (N-hydroxy-4-5-4-(5- Comparative Example 3-2 isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxylpentoxy Group in which N-hydroxy-4-5-4-(5-isopropyl-2- benzamidine (CompoundA) alone was added), the number of methyl-1,3-thiazol-4-yl)phenoxylpentoxy-benzami osteoclast-like cells significantly decreased (Comparative dine (Compound A) at 107M alone was added Example 2-2; 50%) as compared with that in Comparative Example 2-1 (the control group in which the solvent was Comparative Example 3-3 added). In Comparative Example 2-3 (in which risedronic acid alone was added), however, the number of osteoclast Group in which clodronic acid at 10 Malone was like cells showed a tendency to decrease (Comparative added Example 2-3; 88.1%) as compared with that in Comparative Example 2-1 (the control group in which the solvent was Example 3 added), with no significant difference. In Example 2 (in which Group in which N-hydroxy-4-5-4-(5-isopropyl-2-methyl N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl) 1.3-thiazol-4-yl)phenoxylpentoxy-benzamidine (Com phenoxylpentoxy-benzamidine (Compound A) and pound A) at 107M and clodronic acid at 10 Mwere added risedronic acid were added simultaneously), the number of simultaneously osteoclast-like cells remarkably decreased (10.8%) as com pared with those in Comparative Example 2-2 and Compara 0048. The results are shown in FIG. 3. In the graph, the tive Example 2-3, with a significantly lower value as com percentages of the number of osteoclast-like cells formed in pared with that in Comparative Example 2-1 or Comparative each of the Comparative Examples and Example, taking the Example 2-3. In other words, when the above two agents were number of osteoclast-like cells formed in Comparative used in combination, a significantly stronger effect was Example 3-1 as 100%, are shown using the values shown as observed than the effect expected from the sum of the effects the average itstandard deviation of 4 to 5 wells for each group. of the respective single agents. In the Figure, * shows p-0.05 In Comparative Example 3-2 (in which N-hydroxy-4-5-4- as compared with the control group by the Dunnett's t-test; (5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxylpentoxy}- *** shows p-0.001 as compared with the control group by the benzamidine (Compound A) alone was added), the number of Dunnett's t-test; it shows p-0.05 as compared with Compara osteoclast-like cells showed a tendency to decrease (Com US 2010/0216747 A1 Aug. 26, 2010 parative Example 3-2: 95.2%) as compared with that in Com 4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxypen parative Example 3-1 (control group in which the solvent was toxy-benzamidine (described as Compound A in Table 4) added), with no significant difference. In Comparative alone (Comparative Example 4-2), risedronic acid alone Example 3-3 (in which clodronic acid alone was added), (Comparative Example 4-3), and N-hydroxy-4-5-4-(5-iso however, the number of osteoclast-like cells showed a ten propyl-2-methyl-1,3-thiazol-4-yl)phenoxylpentoxy-benza dency to decrease (Comparative Example 3-3; 93.9%) as midine (described as Compound A in Table 4) and risedronic compared with that in Comparative Example 3-1 (control acid (Example 4) were simultaneously added to the mouse group in which the solvent was added), with no significant bone marrow-derived cell culture system, and the effects on difference. In Example 3 (in which N-hydroxy-4-5-4-(5- formation of osteoclast-like cells were evaluated. The follow isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxylpentoxy ing groups were set for the experiment. benzamidine (Compound A) and risedronic acid were added simultaneously), the number of osteoclast-like cells Comparative Example 4-1 decreased markedly (57.3%) as compared with those in Com Control Group (Control) parative Example 3-2 and Comparative Example 3-3, and was significantly lower than those in Comparative Example 3-1, Comparative Example 4-2 Comparative Example 3-2 and Comparative Example 3-3. In Group in which N-hydroxy-4-5-4-(5-isopropyl-2- other words, when the above two agents were used in com methyl-1,3-thiazol-4-yl)phenoxylpentoxy-benzami bination, a significantly stronger effect was observed than the dine (Compound A) at 107M alone was added effect expected from the sum of the effects of the respective single agents. Comparative Example 4-3 0049. In the Figure, * shows p-0.05 as compared with the Group in which Risedronic Acid at 10 MAlone control group by the Dunnett's t-test; it shows p-0.05 as was Added compared with Comparative Example 3-2 by the Student's t-test; and # shows p-0.01 as compared with Comparative Example 4 Example 3-3 by the Student's t-test. Group in which N-hydroxy-4-5-4-(5-isopropyl-2-methyl 0050. The osteoclast-like cell formation inhibition rates of 1.3-thiazol-4-yl)phenoxylpentoxy-benzamidine (Com the respective groups with respect to Comparative Example poundA) at 107M and risedronic acid at 10M were added 3-1 are shown in Table 3. simultaneously 0052 For addition of N-hydroxy-4-5-4-(5-isopropyl-2- TABLE 3 methyl-1,3-thiazol-4-yl)phenoxylpentoxy-benzamidine Effects of N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3-thiazol (Compound A), in both the Comparative Examples and 4-yl)phenoxypentoxy-benzamidine and clodronic acid Example, N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3- on the formation of osteoclast-like cells thiazol-4-yl)phenoxylpentoxy-benzamidine (Compound A) Osteoclast-like cell formation was dissolved in dimethylsulfoxide (DMSO) at a concentra inhibition rate (%) tion of 10 M, and the resultant solution was added to the medium to make 107M at a 1000-fold dilution. For addition Comparative O.O Example 3-1 of risedronic acid, in both the Comparative Examples and Comparative 4.8 Example, sodium risedronate hydrate was dissolved in phos Example 3-2 phate buffered saline (PBS) at a concentration of 10M, and Comparative 6.1 the resultant solution was added to the medium to make 10' Example 3-3 Mata 1000-fold dilution. On every 3 to 4 day after addition Example 3 42.7 of the test compound(s) of the respective conditions, the medium was replaced with fresh 10% FCS-OMEM contain ing the test compound(s) of the respective conditions in both Example 4 the Comparative Examples and Example. Study of the effects of N-hydroxy-4-5-4-(5-isopro 0053 Both in Comparative Examples and Example, the pyl-2-methyl-1,3-thiazol-4-yl)phenoxylpentoxy culture medium was collected 9 days after the start of culture, benzamidine and risedronic acid on the bone resorp and the quantity of type I collagen cross-linked C-telopeptide tion activity of osteoclast-like cells in the medium was measured. Crosslaps for Culture ELISA (Nordic Bioscience Diagnostics, No. 6CRL4000) was used 0051 Multinuclear osteoclast-like cells were formed from for the measurement. Type I collagen cross-linked C-telopep mouse bone marrow-derived cells by collecting bone mar tide is a peptide fragment produced by degradation of ivory by rows of the femur and the tibia from a mouse, removing the bone resorption activity of osteoclast-like cells. erythrocytes according to the ordinary method, plating the 0054. The results are shown in Table 4. In Table 4, the residual components in a 96-well plate containing an ivory inhibitory rate is shown by the percentage of the quantity of section at the density of 4x10 cells/well, culturing the cells type I collagen cross-linked C-telopeptide produced in each overnight, then adding a medium containing 1C,25-dihy group with respect to that in Comparative Example 4-1 using droxy vitamin D (referred to as 1C.25(OH),D, hereinbelow) the average values of 3 to 4 wells for each group. In Com in order to be at 10M, and culturing the cells in the presence parative Example 4-2 (in which N-hydroxy-4-5-4-(5-iso of 10.25(OH), D for 9 days. An CMEM medium containing propyl-2-methyl-1,3-thiazol-4-yl)phenoxylpentoxy-benza 10% fetal calf serum (referred to as 10% FCS-OMEM here midine (Compound A) alone was added) and Comparative inbelow) was used as a medium. When 1C.25(OH), D was Example 4-3 (in which risedronic acid alone was added), the added to the mouse bone marrow-derived cells, each of a number of osteoclast-like cells showed a tendency to decrease solvent alone (Comparative Example 4-1), N-hydroxy-4-5- (Comparative Example 4-2: 13.9%, Comparative Example US 2010/0216747 A1 Aug. 26, 2010

4-3; 18.32%) as compared with that in Comparative Example from the group consisting of etidronic acid, clodronic acid, 4-1 (control group in which the solvent was added), with no pamidronic acid, tiludronic acid, risedronic acid, minodronic significant difference. acid, ibandronic acid, Zoledronic acid, and salts thereof in combination. TABLE 4 1. A preventive ortherapeutic agent for diseases caused by Effects of N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3-thiazol abnormal bone metabolism, comprising as active ingredients 4-yl)phenoxypentoxy-benzamidine and risedronic acid the following (a) and (b): on the bone resorption activity of osteoclast-like cells (a) N-hydroxy-4-5-4-(5-isopropyl-2-methyl-1,3-thia Bone resorption Zol-4-yl)phenoxylpentoxy-benzamidine, or a salt inhibitory rate (%) thereof, and (b) at least one compound selected from the group consist Comparative O.O Example 4-1 ing of etidronic acid, clodronic acid, pamidronic acid, Comparative 13.9 tiludronic acid, risedronic acid, minodronic acid, iban Example 4-2 dronic acid, Zoledronic acid, and salts thereof. Comparative 18.3 Example 4-3 2. The preventive or therapeutic agent for diseases caused Example 4 38.7 (*) by abnormal bone metabolism according to claim 1, wherein the diseases caused by abnormal bone metabolism are dis eases caused by abnormal bone resorption. 0.055 As shown in Table 4, the number of osteoclasts in 3. The preventive or therapeutic agent for diseases caused Example 4 (in which N-hydroxy-4-5-4-(5-isopropyl-2-me by abnormal bone metabolism according to claim 1, wherein thyl-1,3-thiazol-4-yl)phenoxylpentoxy-benzamidine the disease caused by abnormal bone metabolism is (Compound A) and risedronic acid were added simulta osteoporosis. neously) decreased remarkably (38.7%) as compared with 4. The preventive or therapeutic agent for diseases caused those in Comparative Example 4-2 and Comparative by abnormal bone metabolism according to claim 1, wherein Example 4-3, and was significantly lower than that in Com (b) is clodronic acid or risedronic acid or a salt thereof. parative Example 4-1. In other words, when the above two 5. The preventive or therapeutic agent for diseases caused agents were used in combination, a significantly stronger by abnormal bone metabolism according to claim 1, wherein effect was observed than the effect expected from the sum of (b) is risedronic acid or a salt thereof the effects of the respective single agents. 6. The preventive or therapeutic agent for diseases caused 0056. In the Figure, * shows p<0.05 as compared with by abnormal bone metabolism according to claim 1, wherein Comparative Example 4-1 by the Dunnett's t-test. (a) and (b) form a combination drug. 7. The preventive or therapeutic agent for diseases caused INDUSTRIAL APPLICABILITY by abnormal bone metabolism according to claim 1, wherein 0057 The present invention can be used as a preventive or (a) and (b) each are independent single drugs. therapeutic drug or a preventive or therapeutic method for 8. The preventive or therapeutic agent for diseases caused diseases caused by abnormal bone metabolism, especially by abnormal bone metabolism according to claim 1, wherein osteoporosis by administration of N-hydroxy-4-5-4-(5-iso propyl-2-methyl-1,3-thiazol-4-yl)phenoxylpentoxy-benza (a) and (b) are contained in a kit. midine, or a salt thereof and at least one compound selected c c c c c