AU2001284657B2

AU2001284657B2 - Use of vitamin d derivatives as bone resorption inhibitors

Info

Publication number: AU2001284657B2
Application number: AU2001284657A
Authority: AU
Inventors: Atsushi Imaizumi; Seiichi Ishizuka; Noriyoshi Kurihara; Yasuhiro Oue; Sakamuri V. Reddy; G. David Roodman; Kazuya Takenouchi
Original assignee: Teijin Ltd; University of Texas System
Current assignee: Teijin Ltd; University of Texas System
Priority date: 2000-08-23
Filing date: 2001-08-22
Publication date: 2006-01-19
Anticipated expiration: 2021-08-22
Also published as: EP1311253A2; WO2002015894A3; JP2002069003A; WO2002015894A2; NO20030820L; NZ524305A; CA2420274A1; NO20030820D0; AU8465701A

Description

WO 02/15894 PCT/US01/22614 1

DESCRIPTION

USE OF VITAMIN D DERIVATIVES AS BONE RESORPTION INHIBITORS Technical Field The present invention relates to a group of therapeutic agents containing vitamin D antagonists as their active moiety that inhibits bone resorption.

Specifically, the present invention relates to the use of these agents as therapeutic modalities for Paget's disease of bone.

Background Art Bone is a dynamic tissue that undergoes cycles of resorption followed by new bone formation. This process is continued throughout the skeleton in discreet multicellular units called the bone remodeling unit. In the adult bone resorption followed by bone formation is called bone remodeling, and through the remodeling process, bone mass is either maintained or it decreases or increases during life. The relative bone mass depends on the balance between the levels of bone resorption and bone formation. In metabolic bone diseases, this balance between bone resorption and bone formation is changed.

Examples include osteoporosis and Paget's disease of bone.

Osteoporosis is a disease in which both the organic components and mineral of bone decrease. In osteoporosis, bone mass decreases, and fractures of bone are more likely to occur. Osteoporosis has been classified into two types; type I, which is, found in postmenopausal women, and type II, which is found in aged persons. In postmenopausal osteoporosis, also called Type I osteoporosis, estrogen deficiency results in increased local levels of cytokines such as interleukin- 6, interleukin-l1 and interleukin-1. Treatments for WO 02/15894 PCT/US01/22614 2 postmenopausal osteoporosis include, for example, administration of estrogen or its derivatives, bisphosphonates and calcitonin to block-off osteoclastic bone resorption. Similarly, bone metabolism turnover activators such as la-hydroxyvitamin D, and hydroxyvitamin D, preparations derivatives, and bone formation enhancers such as vitamin K 2 preparations, have all been used to treat this disease.

Paget's disease of bone is the most exaggerated example of abnormal bone remodeling. In Paget's disease of bone, bone resorption is markedly increased, followed by abundant new bone formation. The bone laid down is disorganized in structure and results in weakened bone that can bend and fracture. The primary cellular abnormality in Paget's disease of bone resides in the osteoclast, the bone resorbing cell. In Paget's disease of bone, the osteoclast contains paramyxoviral-like nuclei inclusions, which suggests that Paget's disease of bone may be a slow disease caused by paramyxovirus. In addition, there may be a genetic component to Paget's disease of bone. Up to 40% of these patients have a first degree relative affected with the disease. Paget's disease of bone is the second most common bone disease after osteoporosis and it affects up to 2 to 3 million patients in the United States. Paget's disease of bone is normally treated with bisphosphonate or calcitonin, agents that inhibit osteoclast activity and bone resorption. However, recent findings have suggested there are also abnormalities in la,25-dihydroxyvitamin D 3 sensitivity of osteoclast precursors in patients with Paget's disease of bone Bone Miner. Res. vol. 228-236 (2000)). The present inventors have shown that osteoclast precursors from patients with Paget's disease of bone form osteoclasts at concentrations of dihydroxyvitamin D, that are 1 to 2 logs less than (10 to 100 times less than) that are required for normal WO 02/15894 PCT/US01/22614 3 osteoclast formation. Furthermore, these studies have shown that increased sensitivity

D

3 appears to be mediated through induction of a coactivator of the vitamin D receptor. Compounds that interfere with la,25-dihydroxyvitamin D, binding to its receptor, or the effects of la,25-dihydroxyvitamin D 3 on osteoclast precursors, would be a novel and potentially useful agents in treating Paget's disease of bone. If blood levels of la,25-dihydroxyvitamin D 3 in Paget's disease of bone patients are similar to normals, these levels of la,25-dihydroxyvitamin D 3 may be sufficient to induce bone resorption that would not occur normally.

Thus, a pharmaceutical agent that blocks the effects of

D

3 or the hypersensitive osteoclast precursors in Paget's disease of bone, will be very effective therapeutic modalities. Such an agent would have advantages over current therapies for Paget's disease of bone. For example, bisphosphonates in oral form have significant gastrointestinal side effects, and calcitonin therapeutic effectiveness eventually fails over time.

The vitamin D 3 antagonists of the present invention can be synthesized by a method described in the description of international patent publications WO 95/33716 (Compounds of formula WO 00/24712 (Compounds of formula WO 94/07853 (Compounds of formula and WO 97/00242 (Compounds of formula Compounds of formula directly suppress the effects of

D

3 by inhibiting the binding between la,25-dihydroxyvitamin D, and a dihydroxyvitamin D, receptor (VDR) Biol. Chem., vol.

274, 16392-16399 (1999)), the binding between VDR and a the 9-cis-retinoic acid receptor (RXR), and the binding between VDR and a steroid receptor co-activator 1 (SRC-1) of a transcriptional regulation factor Biol. Chem., P\OPER\PDB\Spcci0200)84657 Ispa.doc-2211/ o -4- 0 Z vol. 274, 32376-32381 (1999). Compounds of formula (2) CI appear to antagonize the action of la,25-dihydroxyvitamin D 3 See and J. Biol. Chem., vol. 275, 16506-16512 (2000)).

5 Disclosure of Invention 00 Inventors of the present invention have found that the 0 vitamin D antagonists strongly suppress osteoclast formation O induced by la,25-dihydroxyvitamin D 3 by normal bone marrow cells and bone marrow cells from patients with Paget's disease of bone. This decrease in osteoclast formation results in decreased bone resorption. The inventors have also found that vitamin D antagonists suppress bone resorption induced by la,25-dihydroxyvitamin D 3 in vitamin D deficient animals, demonstrating that these agents have high suppressive effects on bone resorption both in vitro and in vivo.

Advantageously, embodiments of the present invention use vitamin D antagonists to suppress bone resorption patients with Paget's disease of bone. These agents should suppress bone resorption without elevating serum calcium levels.

Thus, in a first aspect there is provided a method of inhibiting bone resorption, comprising administering to a patient a vitamin D antagonist.

The invention further provides in another aspect a use of a vitamin D antagonist for production of a medicament for inhibiting bone resorption.

P:X0PERPDB\Sp=i\2OO 284657 Ispa.doc2111 U03S 4A z Best Mode for Carrying Out the Invention c-i Samples of the vitamin D antagonist of the present invention include compounds expressed by the following formula 00 f (1) WO 02/15894 PCT/US01/22614 5 [in formula m is an integer selected from 1 to 3; q is an integer selected from 0 to 3; r is an integer selected from 0 to 3; X is carbon or oxygen; and 1 q+r S3] Among them, a compound whose m is 1 or 2 is preferable. Further, regarding the combinations of m, q, r and X, compounds shown in Table 1 are preferable; and among them, compounds No. 11, 13, 16, 21, 23 and 26 are especially preferable. In the compounds shown in Table 1, if an asymmetric carbon is present in the structure, it includes both the and configurations.

WO 02/15894 PCT/US01/22614 6 Table 1.

Compound No. m q r X 11 1 0 1 oxygen 12 1 1 1 oxygen 13 1 0 1 carbon 14 1 0 2 carbon 1 0 3 carbon 16 1 1 0 carbon 17 1 2 0 carbon 18 1 3 0 carbon 21 2 0 1 oxygen 22 2 1 1 oxygen 23 2 0 1 carbon 24 2 0 2 carbon 2 0 3 carbon 26 2 1 0 carbon 27 2 2 0 carbon 28 2 3 0 carbon In addition, samples of the vitamin D antagonist of the present invention include compounds expressed by the following formula WO 02/15894 PCT/US01/22614 7

OH

R

3 0

R

4

R

2 HO'' OH (2) [in formula R 1 and R 2 are each hydrogen or they together form an exocyclic methylene; R 3 is a single bond, methylene or vinylene; R 4 is a normal or branched Ci to C, alkyl, alkenyl, alkoxy or alkylamino; R 5 is hydrogen or methyl].

Among them, compounds shown in Table 2 are preferable. In the compounds shown in Table 2, if an asymmetric carbon is present in the structure, the compounds include both the and configurations.

When R 3 is vinylene, the configuration of the double bond includes both (E)-configuration and (Z)-configuration.

P.\OPER\PDB\SpecO001284657 lspadoc-2 /I 1/05 -8- Table 2.

R

3 0

R

Compound No. R R' R3 R 31 exocyclic methylene single bond n-butoxy hydrogen 32 exocyclic methylene single bond 2-methylpropoxy hydrogen 41 exocyclic methylene single bond n-butyl hydrogen 42 exocyclic methylene single bond n-pentyl hydrogen 43 exocyclic methylene single bond n-heptyl hydrogen 44 exocyclic methylene single bond 1-pentenyl hydrogen 51 exocyclic methylene single bond n-butylamino hydrogen 52 exocyclic methylene single bond n-pentylamino hydrogen 53 exocyclic methylene single bond n-heptylamino hydrogen 54 exocyclic methylene single bond 1-pentenylamino hydrogen 61 exocyclic methylene vinylene ethoxy hydrogen 62 exocyclic methylene vinylene t-butoxy hydrogen 71 hydrogen hydrogen single bond n-butoxy hydrogen 81 hydrogen hydrogen vinylene ethoxy hydrogen Advantageously, one or more embodiments of the present invention may inhibit bone resorption using a vitamin D antagonist without elevating the serum calcium concentration. In other embodiments compositions of the present invention may be used for the treatment of a Paget's disease of bone patient, whose bone resorption activity is extremely accelerated by the action of dihydroxyvitamin D 3 Inventors of the present invention found that the vitamin D antagonist of the present invention strongly suppresses formation of osteoclasts induced by WO 02/15894 PCT/US01/22614 9 dihydroxyvitamin D 3 from bone marrow cells of Paget's disease of bone patients, and suppresses the bone resorption induced by la,25-dihydroxyvitamin D 3 in a vitamin D deficient rats.

A bone resorption inhibitor for treating Paget's disease of bone, which contains an above-mentioned compound as the active ingredient, can be formulated as an oral preparation (soft capsules, hard capsules, tablets or a syrup), or in an injectable form with an appropriate vehicle. For example, for treating patients with osteoporosis, an oral preparation may be adequate, but in patients with Paget's disease of bone, who have markedly increased bone resorption, an injectable formulation may be preferable. The injectable formulation should have greater bioavailability.

A vehicle for a parenteral preparation used in the present invention, for example, would be a plant oil, a mineral oil, a white petrolatum, a branched fat or fatand-oil, a high polymeric alcohol or the like. Among these, a plant oil such as cottonseed oil, corn oil, coconut oil or almond oil is preferable. Oils that contain a medium chain fatty acid as a part of the triglyceride is preferred.

Preferred examples of a vehicle for an oral preparation include cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose or methyl cellulose, polyvinyl pyrrolidone, dextrin, cyclodextrin, casein, lactose, mannitol, gelatin or the like.

The amount of an active ingredient for suppressing bone resorption in the present invention is individually decided, depending on the activity of disease, but generally speaking, the amount of an active ingredient is 0.00004 to 0.2 preferably 0.0001 to 0.1 wt.%.

The dosage of the active ingredient is also decided depending on the condition of a patient, but generally WO 02/15894 PCT/US01/22614 10 speaking, it is 0.1 to 1000 pg/day, preferably about 1 to 100 p.g/day. The frequency of administration is commonly 1 to 3 times/day. A preparation is preferably formulated in such a manner that these conditions are satisfied.

Examples The present invention will be explained further in detail hereafter with examples; however, it is not restricted by the examples. Further, the Compound No. in each example is the Compound No. shown in the above Table 1.

Example 1.

Osteoclast formation-suppressing activity of (23S)-25dehydro-la,25-dihydroxyvitamin D3-26, 23-lactone [compound 11. (23S isomer)l on the osteoclast formation induced by la,25-dihvdroxvvitamin D3 from bone marrows cells of normal Persons Mononuclear cells were fractionated from bone marrow cells of healthy normal persons according to a method of Kurihara et al. (Endocrinology, vol. 126, 2733-2741 (1990)). Briefly, bone marrow cells were obtained from normal persons, a mononuclear cell fraction was collected by Hypaque-Ficoll density gradient centrifugation, the cell fraction was washed with a-Minimal Essential Media (a-MEM) 3 times, and the cells dispersed in a-MEM containing 10% fetal bovine serum. This mononuclear cell suspension was seeded in 100-mm tissue culture plates, the culture plate was kept for 90 minutes at 37 0 C in a 4% CO,-air atmosphere, and the non-adherent cells were collected. The non-adherent mononuclear cells were dispersed into a-MEM culture medium containing 20% horse serum at 10 6 cells/ml, the cell suspension was seeded into 96-well multi plates at 100 tl/well.

The capacity of the compound 11 (23S isomer) to WO 02/15894 PCT/US01/22614 11 inhibit osteoclast formation induced by dihydroxyvitamin D, was evaluated as follows: Various concentrations of la,25-dihydroxyvitamin D 3 various concentrations of compound 11 (23S isomer) or a combination of 10 8 M la,25-dihydroxyvitamin D, solution and various concentrations of compound 11 (23S isomer) solution were added to each well. The culture medium was replaced twice weekly and the cultures continued for 3 weeks at 37 0 C in an atmosphere of 4% CO-air.

Osteoclasts that formed were identified by their capacity to bind the 23C6 antibody. The nuclei were counter stained with methyl green. Cells that reacted with the 23C6 antibody and had 3 or more nuclei were scored as osteoclasts. The results are shown in Table 3.

Table 3.

Compound 11 (23S isomer) inhibit osteoclast formation induced by la,25-dihydroxvvitamin D3 in normal human bone marrow cultures.

compound concentration control (without adding test compounds) 1a,25-dihydroxyvitamin D 3 10 1 1

M

1010 M 9

M

10-8 M 7

M

compound 11 (23S isomer) 10 11

M

1 0 M 9

M

8

M

7

M

6

M

D

3 10-8 M compound 11 (23S isomer) 10-1 M compound 11 (23S isomer) 10 10

M

compound 11 (23S isomer) 10- 9

M

compound 11 (23S isomer) 10 8

M

compound 11 (23S isomer) 10- 7

M

compound 11 (23S isomer) 10- 6

M

osteoclast formation (average number of cells S.D.) 17 13 3 11 2 34 6 87 12 91 7 17 4 9 5±1 3 2 0 1 0 0 WO 02/15894 PCT/US01/22614 12

D

3 induced osteoclast formation in a dose-dependent manner at 10 9 M to 10 7 M concentrations of la,25-dihydroxyvitamin D 3 Osteoclast formation was maximal at 10 8 M la,25-dihydroxyvitamin D 3 The compound 11 (23S isomer) did not induce osteoclast formation.

When 10 8 M la,25-dihydroxyvitamin D. and various concentrations of compound 11 (23S isomer) were added simultaneously to the normal marrow cultures, osteoclast formation induced by the la,25-dihydroxyvitamin D, was suppressed by compound 11 (23S isomer) at concentration of 10 9 M to 10 6

M.

Example 2.

Effects of (23S)-25-dehydro-la,25-dihvdroxyvitamin D3-26, 23-lactone [compound 11, (23S isomer)] on osteoclast formation induced by la,25-dihydroxyvitamin D3 in bone marrow cultures from Paget's disease of bone patients.

Bone marrow cells were obtained from involved bones of patients with Paget's disease of bone and processed and cultured as described above for normal bone cells.

The cultures were treated in an identical manner with

D

3 and/or compound 11 (23S isomer) as described in Example 1. The results are shown in Table 4.

WO 02/15894 PCT/USOI/22614 13 Table 4.

Compuound 11 (23S isomer) inhibit osteoclast formation induced by 1a,25-dihydroxvvitamin Ds in bone marrow cultures from Pagiet's disease of bone patients.

compound concentration control (without adding test compounds)

D

3 10-11 M 10-10 M l0-1 M l0-1 M 10-1 M compound 11 (23S isomer) 10-11 M.

10-10 M lo- M 10-1 M 10-7 M 10-1 M

D

3 10-10 M compound 11 (23S isomer) 1C)" M compound 11 (23S isomer) 10-1(1 M compound 11 (23S isomer) 10-9 M "I compound 11 (23S isomer) 1008 M compound 11 (23S isomer) 10-7 M compound 11 (23S isomer) 10-6 M osteoclast formation (average number of cells S.D.) 47 6 134 14 180 211 206 16 205 ±6 29 20 ±4 16 ±2 9±3 8±2 170 ±13 155 ±6 125 63 14 ±1 1±1 In bone marrow cultures from patients with Paget's disease of bone, lat,25-dihydroxyvitamin D, induced osteoclast formation at concentrations as low as 10-31 M.

lci,25-dihydroxyvitamin D 3 induced osteoclast formation in these cultures in a dose-dependent fashion from 10'1 M to 10-7 M. Osteoclast formation was maximally in these cultures between 10 0 M and 10-' M. These are dihydroxyvitamin D 3 concentrations that are 1 to 2 logs less than that are required for maximal osteoclast formation in normal marrow cultures. Compound 11 (23S isomer) by itself did not induce osteoclast formation and inhibited basal osteoclast formation in the absence of 1c',25-dihydroxyvitamin D 3 or when D3was added. Furthermore, when the 10-:0 M dihydroxyvitamin D. was added to these cultures in WO 02/15894 PCT/US01/22614 14 combination with varying concentrations of compound 11 (23S isomer) osteoclast formation was inhibited in dosedependent fashion between 10 11 M and 10 6 M compound 11 (23S isomer) concentrations. Note that compound 11 (23S isomer) inhibited the increased basal osteoclast formation in cultures of Paget's disease of bone patients, which was not seen in cultures from normal marrow. These data suggest that at physiologic concentrations of la,25-dihydroxyvitamin D 3 compound 11 (23S isomer) may return osteoclast formation to normal levels in patients with Paget's disease of bone.

Example 3.

Osteoclast inhibitory capacity of different types of vitamin D antagonists on osteoclast formation induced by D3 from bone marrow cultures of Paget's disease of bone patients.

Bone marrow cultures were performed as described in Example 1, with the exception that different types of vitamin D antagonists were added to at varying concentrations to bone marrow cultures stimulated with

D

3 The bone marrow cells were taken from involved bones of patients with Paget's disease of bone and the cultures were performed as described in Example 2. There are two diastereoisomers, based on the asymmetric carbon at the 23 position, in compounds 13 and 16. The isomer having the shorter retention time on reverse phase HPLC analysis was the more polar isomer and the isomer having the longer retention time was the less polar isomer. The results are shown in Table WO 02/15894 PCT/US01/22614 15 Table Osteoclast formation-suppressing activities of various vitamin D antagonists on the osteoclast formation induced by la,25-dihydroxyvitamin D3 in bone marrow cultures from Paget's disease of bone patients compound concentration osteoclast formation (average number of cells S.D.) control (without adding test compounds)

D

3 10 1

M

10-10 M 9

M

10-8 M compound 11 (23R isomer) 10 9

M

8

M

7

M

compound 13 (more polar isomer) 10 9

M

8

M

7

M

compound 16 (more polar isomer) 10 9

M

8 M 7

M

compound 13 (less polar isomer) 10- 9

M

10-8 M

M

1 O-i 2 hTr r d xvtamin n, 10-10 M 47 6 134 14 180 211 206 16 32+ 3 20 3 1 1 44 25 4 3 4 15 2 8 3 1 1 7 1 6 2 1 1 comY (2R isomer compound 11 (23R isomer) compound 11 (23R isomer) compound 11 (23R isomer)

D

3 compound 13 (more polar isomer) compound 13 (more polar isomer) compound 13 (morepolar isomer)

D

3 compound 16 (morepolar isomer) compound 16 (more polar isomer) compound 16 (more polar isomer)

D

3 compound 13 (less polar isomer) compound 13 (less polar isomer) compound 13 (less polar isomer) 10-9 M 10- M 10- 7

M

10-10 M 10- 9

M

10-8 M 10- 7

M

10-10 M 10- M 10- 8

M

10- 7

M

10- 1 0

M

10- 9

M

10- 8

M

10 7

M

145 70 18 152 79 31 131 67 18 127 70 14 As shown in Table 5, la,25-dihydroxyvitamin

D,

induced osteoclast formation in a dose-dependently manner from 10- 11 M to 10 8 M. Compound 11 (23R isomer), compound 13 (more polar isomer and less polar isomer) and compound 16 (more polar isomer) alone did not induce the osteoclast formation, but rather inhibited basal WO 02/15894 PCT/US01/22614 16 osteoclast formation. Osteoclast formation induced by 10 M la,25-dihydroxyvitamin D 3 was suppressed by the compound 11 (23R isomer), the compound 13 (more polar isomer or less polar isomer) or compound 16 (more polar isomer) in a dose-dependent manner in the range of 10 9

M

to 10- 7 M. At the concentration of 10 7 M, osteoclast formation induced by 10 10 M la,25-dihydroxyvitamin D 3 was almost completely suppressed by all compounds.

Example 4.

Measurement of vitamin D metabolites in blood for Paget's disease of bone patients and normal persons of a similar age Blood samples (7 to 10 ml) were collected from 9 Paget's disease of bone patients and 10 normal persons of similar ages and the blood samples were held at room temperature for 3 hours. Afterward, they were centrifuged at 3000 rpm for 10 minutes to obtain sera.

The concentrations of vitamin D metabolites in the sera were measured by the method of Ishizuka et al. (Journal of Nutritional Science and Vitaminology, vol. 27, 71-75 (1981) and Acta Endocrinology, vol. 104, 96-102 (1983)).

Briefly, 3 to 5 ml of each serum sample was diluted 1:3 with water, a chloroform:methanol mixed solution in a volume that was two times the volume of the diluted serum was added, and the suspension was vigorously shaken followed by collecting the chloroform layer. The water layer was extracted again with chloroform. The pooled chloroform layers obtained were concentrated on a rotary evaporator, and the residue was placed on a Sephadex LHcolumn (1.2x10 cm) and eluted with a mixed solvent of n-hexane:chloroform:methanol The 8-17 ml eluent and the 19-60 ml eluent were collected as the fraction and the 24,25-(OH) 2 D la,25-(OH) 2 D fraction, respectively. Further, the 25-OH-D fraction and the 24,25-(OH) 2 D la,25-(OH) 2 D fraction were each placed on WO 02/15894 PCT/US01/22614 17 a Zorbax SIL column (4.6x250 mm) and eluted with 12% isopropanol in n-hexane to purify the 25-OH-D, 24,25-

(OH)

2 D and la,25-(OH) 2 D fractions. The concentrations of the purified 25-OH-D fraction and 24,25-(OH),D fraction were determined by a competitive protein binding assay, using vitamin D binding protein present in serum from a vitamin D deficient rat. The concentration of the purified la,25-(OH) 2 D was determined by a radioreceptor assay, using the vitamin D receptor from the small intestine of a vitamin D deficient chick. The results are shown in Table 6.

Table 6.

Measurement of vitamin D metabolites in blood from Paget's disease of bone patients and normal persons of similar age serum concentration of vitamin D metabolite sample 25-OH-D 24,25-(OH) 2 D la,25-(OH) 2

D

(ng/ml) (ng/ml) (pg/ml) Paget's patients 40.5±11.1 2.64±1.57 41.0±9.1 Normals 39.3±9.5 2.39±1.09 38.8±12.0 In the Table 6, the data are expressed as mean value±S.D.

The concentrations of vitamin D metabolites in sera from Paget's disease of bone patients did not significantly differ from normals of the similar age.

Abnormalities of vitamin D metabolism were not detected in Paget's disease of bone patients. These data show that the concentration of la,25-(OH),D in sera from Paget's disease of bone patients was 41.0±9.1 pg/ml serum 1 0

M).

As is clear from Examples 1 and 2, osteoclast formation by bone marrow cells from Paget's disease of bone patients, is induced by 10 10 M dihydroxyvitamin

D

3 In contrast, osteoclast formation WO 02/15894 PCT/US01/22614 18 from bone marrow cells of normal persons, is not induced by 10-10 M la,25-dihydroxyvitamin D 3 Suda et al. have shown that there is a positive correlation between osteoclast formation and bone resorption (Suda, T and Takahashi, N, Endocrine Review, vol. 20, 345-357 (1999)).

That is, compounds stimulating osteoclast formation also affect bone resorption activity.

Example Bone resorption-suppressing activity of (23S)-25-dehydro- D3-26, 23-lactone [compound 11, (23S isomerl] on the bone resorption induced by dihydroxyvitamin D3 in vitamin D deficient rats Four-week-old male Wistar rats purchased from Japan SLC (Shizuoka, in Japan) were used. The animals were kept in wire-net cages (three animals in a cage) and raised for 7 weeks with free access to a vitamin Ddeficient diet (Ca, 0.0036%; P, Harlan Taklad Research Diet, Madison, WI, and drinking water (well water treated with hypochlorite of 0.4%±0.2 ppm).

Temperature was kept at 23±1 0 C and the humidity at 55±10%. Five animals were used for each experimental group. The control animals received intravenously administered solvent ethanol-0.1% Triton X-100physiological saline solution), and the experimental animals were intravenously administered dihydroxyvitamin D, at a dose of 0.25 ig/kg. For animals receiving vitamin D derivatives, compound 11 (23S isomer) was administered intravenously at a dose of 2 pg/kg, p.g/kg or 50 hg/kg, or the compound 11 (23S isomer) with or without la,25-dihydroxyvitamin D, at a dose of 0.25 g/kg. The volume was 2 ml/kg for each animal. 24 hours after administration, a blood sample was taken out from abdominal descending aorta under ether anesthesia, the WO 02/15894 PCT/US01/22614 19 serum was collected according to conventional methods, and the calcium concentration in the serum was measured.

The calcium concentrations were measured according to OCPC-method (Am. J. Clin. Pathol., vol. 45, 290-296 (1966)) with an autoanalyzer (type 7070, manufactured by Hitachi Seisakusho). The results are shown in Table 7.

Table 7.

Bone resorption-suppressing activity of compound 11 (23S isomer) on serum calcium levels induced by dihydroxyvitamin D3 in vitamin D deficient rats Sd serum Ca concentration compound dose (mg/100 ml serum) control 4.64±0.19

D

3 0.25 g/kg 5.42±0.12** compound 11 (23S isomer) 2 g/kg 4.61±0.12 compound 11 (23S isomer) 10 ig/ kg 4.67±0.11 compound 11 (23S isomer) 50 pg/ kg 4.65±0.18 D3 0.25 pg/kg compound 11 (23S isomer) 2 tg/kg 5.31±0.22 compound 11 (23S isomer) 10 pg/kg 4.97±0.18 a compound 11 (23S isomer) 50 pg/kg 4.72±0.21' The data are expressed as the mean value±S.D.

Significantly different from control: p<0.001.

Significantly different from la,25-(OH),D 3 a, p<0.01, and b, p<0.01.

The results show that 24 hours after receiving 0.25 [g/kg of la,25-dihydroxyvitamin D 3 animals had a significant increased serum calcium concentration compared to controls. Since these rats were raised on a calcium free diet, the increased serum calcium is attributable to the calcium released from bone by osteoclastic bone resorption (Am. J. Physiol., vol. 216, 1351-1359 (1969)). For animals receiving compound 11 (23S isomer), increased serum calcium concentrations were not observed even at a dose of 50 ig/kg. Thus, the

I

P:\OPER\PDB\Spcci\2001284657 Ispa.doc-22 1/05

O

Z compound does not induce bone resorption 24 hours after C administration. However, when la,25-dihydroxyvitamin D 3

(N

(0.25 pg/kg) and compound 11 (23S isomer) were administered simultaneously at a dose of 2 pg/kg, 10 pg/kg or 50 pg/kg, l 5 bone resorption was not increased. These data demonstrate O that bone resorption induced la,25-dihydroxyvitamin D 3 was suppressed by the administration of compound 11 (23S isomer) p in a dose-dependent manner.

These results demonstrate that the vitamin D antagonist of the present invention suppresses bone resorption induced by la,25-dihydroxyvitamin D 3 without increasing serum calcium concentrations in vivo. Thus, the vitamin D antagonist of the present invention is useful for treating the increased bone resorption attributable to la,25-dihydroxyvitamin D 3 that is seen in Paget's disease of bone patients.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims

1. A method of inhibiting bone resorption, O comprising administering to a patient a vitamin D antagonist. c 1 5 2. A method for treating Paget's disease of bone, comprising administering to a patient a vitamin D antagonist. O 3. The method of claim 1, wherein said antagonist 00 has the formula M rt HO' -^OH (1) wherein m is an integer selected from 1 to 3; q is an integer selected from 0 to 3; r is an integer selected from 0 to 3; X is carbon or oxygen; and 1 5 q+r s 3.

4. The method of claim 2, wherein said antagonist has the formula HO'* OH (1) wherein m is an integer selected from 1 to 3; q is an integer selected from 0 to 3; r is an integer selected from 0 to 3; X is carbon or oxygen; and 1 5 q+r 3.

5. The method of claim 3, wherein m is 1 or 2.

6. The method of claim 4, wherein m is 1 or 2.

7. The method of claim 3, wherein m is 1, q is 0, WO 02/15894 PCT/US01/22614 22 r is 1, and X is oxygen.

8. The method of claim 4, r is 1, and X is oxygen

9. The method of claim 3, r is 1, and X is carbon. The method of claim 4, r is 1, and X is carbon.

11. The method of claim 3, r is 1, and X is oxygen.

12. The method of claim 4, r is 1, and X is oxygen.

13. The method of claim 3, r is 0, and X is carbon.

14. The method of claim 4, r is 0, and X is carbon. The method of claim 1, has the formula wherein m is 1, wherein m is 1, q is 0, q is 0, wherein m is 1, q is 0, wherein m is 2, q is 0, wherein m is 2, q is 0, wherein m is 1, q is 1, wherein m is 1, q is 1, wherein said antagonist R R 4 wherein R 1 and R 2 are each hydrogen or they together form an exocyclic methylene; R 3 is a single bond, methylene or vinylene; R 4 is a normal or branched C, to C, alkyl, alkenyl, alkoxy or alkylamino; R 5 is hydrogen or methyl.

16. The method of claim 2, wherein said antagonist has the formula WO 02/15894 PCT/US01/22614 23 R 5 OH R 3 o R4 R R 2 HO"' OH (2) wherein R 1 and R 2 are each hydrogen or they together form an exocyclic methylene; R 3 is a single bond, methylene or vinylene; R 4 is a normal or branched C, to C, alkyl, alkenyl, alkoxy or alkylamino; R 5 is hydrogen or methyl.

17. The method of claim 15, wherein R 4 is n-butyl, n-pentyl, n-hexyl, n-heptyl, 1-pentenyl, methoxy, ethoxy, n-propoxy, iso-propoxy, 2-methylpropoxy, n-butoxy, t- butoxy, n-pentyloxy, n-hexyloxy or n-heptyloxy, n- butylamino, n-pentylamino, n-heptylamino, n- pentenylamino.

18. The method of claim 16, wherein R 4 is n-butyl, n-pentyl, n-hexyl, n-heptyl, 1-pentenyl, methoxy, ethoxy, n-propoxy, iso-propoxy, 2-methylpropoxy, n-butoxy, t- butoxy, n-pentyloxy, n-hexyloxy or n-heptyloxy, n- butylamino, n-pentylamino, n-heptylamino, n- pentenylamino.

19. The method of claim 15, wherein R' and R 2 together form an exocyclic methylene; R 3 is a single bond; R 4 is n-butoxy or 2-methylpropoxy; R 5 is hydrogen. The method of claim 16, wherein R' and R 2 together form an exocyclic methylene; R 3 is a single bond; R 4 is n-butoxy or 2-methylpropoxy; R 5 is hydrogen.

21. The method of claim 15, wherein R' and R 2 together form an exocyclic methylene; R 3 is a single bond; R 4 is n-butyl, n-pentyl, n-heptyl or 1-pentenyl; R is hydrogen.

22. The method of claim 16, wherein R' and R 2 together form an exocyclic methylene; R 3 is a single 24 0 bond; R 4 is n-butyl, n-pentyl, n-heptyl or 1-pentenyl; R is hydrogen. O 23. The method of claim 15, wherein R 1 and R 2 together form an exocyclic methylene; R 3 is a single C( 5 bond; R 4 is n-butylamino, n-pentylamino, n-heptylamino or 1-pentenylamino; R 5 is hydrogen.

24. The method of claim 16, wherein R' and R 2 I n ND together form an exocyclic methylene; R 3 is a single 00 bond; R 4 is n-butylamino, n-pentylamino, n-heptylamino or 1-pentenylamino; R S is hydrogen. The method of claim 15, wherein R' and R 2 C together form an exocyclic methylene; R 3 is vinylene; R 4 is ethoxy or t-butoxy; R 5 is hydrogen.

26. The method of claim 16, wherein R' and R 2 together form an exocyclic methylene; R 3 is vinylene; R 4 is ethoxy or t-butoxy; R 5 is hydrogen.

27. The method of claim 15, wherein R' and R 2 are each hydrogen; R 3 is a single bond; R' is n-butoxy; R 5 is hydrogen.

28. The method of claim 16, wherein R 1 and R 2 are each hydrogen; R 3 is a single bond; R 4 is n-butoxy; R 5 is hydrogen.

29. The method of claim 15, wherein R 1 and R 2 are each hydrogen; R 3 is vinylene; R 4 is ethoxy; R 5 is hydrogen. The method of claim 16, wherein R' and R 2 are each hydrogen; R 3 is vinylene; R 4 is ethoxy; R S is hydrogen. P:\OPER\PDB\Speci\2001284657 Ipa.doc-21/1 1/05

31. A use of a vitamin D antagonist for production of a medicament for inhibiting bone resorption.

32. A use of a vitamin D antagonist for production of a medicament for treating Paget's disease of bone.

33. The use of claim 31, wherein said antagonist has the formula wherein m is an integer selected from 1 to 3; q is an integer selected from 0 to 3; r is an integer selected from 0 to 3; X is carbon or oxygen; and 1 q+r 3.

34. The use of claim 32, wherein said antagonist has the formula (1) wherein m is an integer selected from 1 to 3; q is an integer selected from 0 to 3; r is an integer selected from 0 to 3; X is carbon or oxygen; and 1 q+r 3. The use of claim 31, wherein said antagonist has the formula P:\OPER\PDB\Speii2001284657 Ispa.doc-22/11/05 O In 0D -26- AS OR AR 00 C H v OH (2) wherein R 1 and R 2 are each hydrogen or they together form an exocyclic methylene; R 3 is a single bond, methylene or vinylene; R 4 is a normal or branched Ci to C7 alkyl, alkenyl, alkoxy or alkylamino; R 5 is hydrogen or methyl.

36. The use of claim 32, wherein said antagonist has the formula SR 5 OH I rR1 R HO' OH (2) wherein R 1 and R 2 are each hydrogen or they together form an exocyclic methylene; R 3 is a single bond, methylene or vinylene; R 4 is a normal or branched Ci to C7 alkyl, alkenyl, alkoxy or alkylamino; R 5 is hydrogen or methyl.

37. A method according to claim 1 substantially as hereinbefore described and/or exemplified. m P:\OPER\PDB\SpwiA20W284637 Ispa.dom-2I/1 1/05 -27- O z 38. A use according to claim 31 substantially as c- hereinbefore described and/or exemplified. (N DATED this 2 2 nd day of November, 2005 Teijin Limited AND Board of Regents, The University of Texas System By DAVIES COLLISON CAVE Patent Attorneys for the Applicant