Isoprenoid pathway inhibitors for stimulating bone growth

Details Isoprenoid pathway inhibitors for stimulating bone growth Isoprenoid pathway inhibitors for stimulating bone growth

2000-01-20

2002-04-23

Gitomer, Ralph (Department: 1623)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Phosphorus containing other than solely as part of an...

C514S333000, C514S374000

Reexamination Certificate

active

06376476

ABSTRACT:

TECHNICAL FIELD
The invention relates to screening methods for compositions and to methods to use these compositions in treating bone disorders in vertebrates including fractures and cartilage disorders. More specifically, the invention concerns a method to identify agents that will be useful in treating bone disorders by assessing their ability to inhibit enzymes in the isoprenoid synthesis pathway and in particular, to inhibit HMG-CoA reductase, and to methods and compositions useful in treating bone disorders which contain the identified active ingredients as essential components.
BACKGROUND ART
Bone is subject to constant breakdown and resynthesis in a complex process mediated by osteoblasts, which produce new bone, and ostcoclasts, which destroy bone. The activities of these cells are regulated by a large number of cytokines and growth factors, many of which have now been identified and cloned.
There is a plethora of conditions which are characterized by the need to enhance bone formation. Perhaps the most obvious is the case of bone fractures, where it would be desirable to stimulate bone growth and to hasten and complete bone repair. Agents that enhance bone formation would also be useful in facial reconstruction procedures. Other bone deficit conditions include bone segmental defects, periodontal disease, metastatic bone disease, osteolytic bone disease and conditions where connective tissue repair would be beneficial, such as healing or regeneration of cartilage defects or injury. Also of great significance is the chronic condition of osteoporosis, including age-related osteoporosis and osteoporosis associated with post-menopausal hormone status. Other conditions characterized by the need for bone growth include primary and secondary hyperparathyroidism, disuse osteoporosis, diabetes-related osteoporosis, and glucocorticoid-related osteoporosis.
One group of compounds suggested for enhancing bone formation comprises bone morphogenic proteins (BMPs). The BMPs are novel factors in the extended transforming growth factor &bgr; superfamily. Recombinant BMP-2 and BMP-4 can induce new bone formation when they are injected locally into the subcutaneous tissues of rats (Wozney J.
Molec Reprod Dev
(1992) 32:160-67). These factors are expressed by normal osteoblasts as they differentiate, and have been shown to stimulate osteoblast differentiation and bone nodule formation in vitro as well as bone formation in vivo (Harris S., et al.,
J. Bone Miner Res
(1994) 9:855-63). This latter property suggests potential usefulness as therapeutic agents in diseases which result in bone loss.
The cells which are responsible for forming bone are osteoblasts. As osteoblasts differentiate from precursors to mature bone-forming cells, they express and secrete a number of enzymes and structural proteins of the bone matrix, including Type-I collagen, osteocalcin, osteopontin and alkaline phosphatase (Stein G., et al.,
Curr Opin Cell Biol
(1990) 2:1018-27; Harris S., et al., (1994), supra). They also synthesize a number of growth regulatory peptides which are stored in the bone matrix, and are presumably responsible for normal bone formation. These growth regulatory peptides include the BMPs (Harris S., et al. (1994), supra). In studies of primary cultures of fetal rat calvarial osteoblasts, BMPs 1, 2, 3, 4, and 6 are expressed by cultured cells prior to the formation of mineralized bone nodules (Harris S., et al. (1994), supra). Like alkaline phosphatase, osteocalcin and osteopontin, the BMPs are expressed by cultured osteoblasts as they proliferate and differentiate.
Although the BMPs are potent stimulators of bone formation in vitro and ill vivo, there are disadvantages to their use as therapeutic agents to enhance bone healing. Receptors for the bone morphogenetic proteins have been identified in many tissues, and the BMPs themselves are expressed in a large variety of tissues in specific temporal and spatial patterns. This suggests that BMPs may have effects on many tissues in addition to bone, potentially limiting their usefulness as therapeutic agents when administered systemically. Moreover, since they are peptides, they would have to be administered by injection. These disadvantages impose severe limitations to the development of BMPs as therapeutic agents.
Small molecules that are useful in treating bone disorders in vertebrates are of the general formula Ar
1
—L—Ar
2
wherein Ar
1
and Ar
2
are aromatic moieties and L is a linker that separates them by a specified distance. These arc disclosed in PCT application WO98/17267 published Apr. 30, 1998. These compounds were assessed for usefulness in treating bone disorders by their ability to enhance the production of a reporter protein when the nucleotide sequence encoding the reporter protein is operably linked to the promoter for BMP-2. Similar compounds arc disclosed for this purpose in earlier filed PCT applications WO097/15308 published May 1, 1997 and WO097/48694 published Dec. 24, 1997. The present application concerns another class of compounds that arc inhibitors of &bgr;-hydroxy-&bgr;-methyl glutaric acid CoA (HMG-CoA) reductase that are also successful in this assay. The compounds described in the present application are generically known as “statins.”
Statins are HMG-CoA reductase inhibitors (Bilheimer, D. W.,
Drug Investigation
(1990) 2 (Suppl. 2) 58-67). HMG-CoA reductase is the principal rate limiting enzyme involved in cellular cholesterol biosynthesis. The pathway is also responsible for the production of dolichol, ubiquinones, isopentenyl adenine and farnesol. HMG-CoA reductase converts 3-hydroxy-3-methyl-glutaryl CoA (HMG-CoA) to mevalonate. Addition of mevalonate at concentrations between 25-800 &mgr;M inhibits the activity of mevastatin (100, 25, or 6.25 &mgr;M) in the ABA assay described in Example 1 herein. Mevalonic acid has no effect on primary screen activities of bone growth-active compounds outside of the statin family (compounds 59-0008 (see Example 1)). These data indicate that the effect of mevastatin in the ABA assay is mediated by its effect on HMG-CoA reductase. Knowledge of inhibitors of the cholesterol biosynthetic pathway (including SAR or pharmacophore analyses) may be useful in determining appropriate modifications or analogs of the statins that maintain bone growth activity.
U.S. Pat. No. 5,280,040 discloses compounds described as useful in the treatment of osteoporosis. These compounds putatively achieve this result by preventing bone resorption. Related to these compounds are the bisphosphonates—the methylene bisphosphonic acids. These compounds are comprised of two phosphonic acid residues coupled through a methylene linkage. Typical representatives include the clodronates which are simple compounds wherein the phosphonic acid residues are coupled through dichloromethylene. Other representative bisphosphonates include ibandronates, the risedronates, alandronates and pamidronates. These compounds have been shown to inhibit the resorption of bone, presumably by effecting apoptosis of osteoclasts. Luckman, S. P., et al.,
J Bone Min Res
(1998) 13:581-589.
Wang, G.-J., et al.,
J Formos Med Assoc
(1995) 94:589-592 report that certain lipid clearing agents, exemplified by lovastatin and bezafibrate, were able to inhibit the bone resorption resulting from steroid administration in rabbits. There was no effect oil bone formation by these two compounds in the absence of steroid treatment. The mechanism of the inhibition in bone resorption observed in the presence of steroids (and the mechanism of the effect of steroid on bone per se) is said to be unknown. The authors state that steroid-induced bone loss is associated with a decrease in bone formation attributed to an inhibitory effect of corticosteroid on osteoblast activity and an increase in bone absorption due to direct osteoclast stimulation and to an indirect inhibition of intestinal calcium absorption with a secondary increase in parathyroid hormone production. Other mechanisms mentioned include those attributable to lipid abnormalities

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