Suppression, by 5-lipoxygenase inhibitors, of bone resorption

Details Suppression, by 5-lipoxygenase inhibitors, of bone resorption Suppression, by 5-lipoxygenase inhibitors, of bone resorption

2000-05-08

2002-09-24

Criares, Theodore J. (Department: 1617)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Having -c-, wherein x is chalcogen, bonded directly to...

C514S418000, C514S419000, C514S451000, C514S734000, C514S736000, C514S259500, C514S311000, C514S307000, C514S453000, C514S460000

Reexamination Certificate

active

06455541

ABSTRACT:

BACKGROUND OF THE INVENTION
Diseases associated with bone loss are usually accompanied by increased osteoclast activation. Such diseases include estrogen deficiency after the menopause osteoporosis, primary hyperparathyroidism, malignancy, Paget's disease of bone and periodontal disease. The bone loss is caused by osteoclast activity. Osteoclasts are unique multinucleated cells within bone that are responsible for bone degradation. These are the only cells in the body known to be capable of resorbing bone. Since diseases of bone loss are associated with increased activity of these cells, it is important to understand the mechanisms by which osteoclasts are activated in these disease states, and to devise rational and therapeutic means to inhibit this activation.
The molecular mechanisms by which osteoclasts are activated are unknown. In vitro data indicate cytokines and systemic hormones with bone resorbing effects do not act directly on osteoclasts, but rather act on accessory cells in the bone marrow microenvironment and that these cells in turn are responsible for osteoclast activation (Rodan & Martin 1981, Mcsheehy & Chambers 1986). This activation may be mediated either by cell-cell contact or by locally active soluble factors. In a search for cell sources of such soluble factors, the present inventors found that a stromal cell line (C433) derived from a giant cell tumor of bone produced prodigious amounts of osteoclast-stimulating activity greater than any we found in conditioned media from cells with osteoblast characteristics (Oreffo et al., 1993).
Human giant cell tumors of bone comprise heterogeneous cell populations, including giant cells with many of the phenotypic and functional characteristics of osteoclasts as well as mononuclear cells. The multinucleated cells are positive for osteoclast surface antigens (Davies et al, 1989), for tartrate-resistant acid phosphatase (TRAP),
1
possess receptors for calcitonin (Komiya et al., 1990) and lack monocyte-macrophage surface antigens (Goldring et al., 1986). The mononuclear cells comprise two distinct populations. One population does not persist in culture and is positive for Ia and monocyte-macrophage antigens (Ling et al., 1988). Another population persists in culture and resembles connective tissue stromal cells, produces Types I and III collagen, and has receptors for parathyroid hormone (Goldring et al., 1986). These latter cells can be readily established in cell culture. One cell line (C433) derived from stomal cells from a giant cell tumor is shown herein to cause greater increases in osteoclast activity as measured by accumulation of TRAP activity than any of the known osteoblast-like cell lines. This study concerns characterizing the osteoclast stimulating activity produced by this cell line. This activity is ascribed to 5-hydroxyeicosanoids, which are 5-lipoxygenase metabolites of arachidonic acid.
1
The abbreviations used include: TRAP, tartrate-resistant acid phosphatase; HETE, hydroxyeicosatetraenoic acid; HPLC, high pressure measure liquid chromatography; LTC
4
, LTD
4
, and LTE
4
, leukotriene C
4
, leukotriene D
4
, and leukotriene E
4
; GC-MS, gas chromatography-mass spectrometry, 5-LO, 5-lipoxygenase.
Dziak and co-workers (Mohammed et al., 1989) examined the role of leukotrienes in orthodontic tooth movement, a model used to examine bone remodeling. These investigators found significant inhibition of tooth movement using the leukotriene inhibitor AA 861, even though enhanced levels of prostaglandins were detected in this treated tissue. They suggested that inhibition of LT synthesis might influence tooth movement and that prostaglandins and leukotrienes might mediate different steps in a cascade of events that results in initiation of bone remodeling.
5-lipoxygenase metabolites possess a diverse range of biological activities, especially in allergic and inflammatory responses. The molecule LTB-4 is chemotactic for polymorphonuclear leukocytes, eosinophils, lymphocytes, and monocytes and will increase adherence, oxygen radical production, and lysosomal degranulation in polymorphonuclear leukocytes (Goldman et al., 1986). LTC-4 and LTD-4 have been shown to promote myeloid colony formation (Ziboh et al., 1986), proliferation of glomerular epithelial cells (Baud et al., 1985), and secretion of luteinizing hormone and luteinizing hormone releasing-hormone (Parker 1987). A number of cytokines and growth factors such as interleukin-1 and interleukin-2 will induce production and secretion of leukotrienes (Parker 1987). Significant breakthroughs have been made in identifying proteins and enzymes involved in the synthesis of these compounds. For example, only cells which contain both 5-lipoxygenase (5-LO) and a recently cloned protein, 5-lipoxygenase-activating protein (FLAP), will produce leukotrienes (Dixon et al., 1990; Reid et al., 1990). Hormones which induce 5-LO metabolite production may regulate not only 5-LO enzymes but FLAP expression.
There are previous reports that leukotrienes (LT) may be modulators of bone cell function. Meghji et al (1988) tested purified leukotrienes in the neonatal mouse calvarial assay and found significant bone resorption. However, other investigators have not been able to repeat these results (personal communications). This may be due to the unstable nature of these compounds. The present inventors now report that special precautions are necessary to maintain biological activity. The compounds must be stored under argon in the absence of light. Once removed from these conditions the commercial compounds must be diluted quickly and used immediately. Activity of commercially available compounds was detectable at higher concentrations (10
−6
to 10
−7
M) in the organ culture assay, whereas activity was detectable at much lower concentrations (10
−10
to 10
−11
M) in the isolated avian osteoclast and isolated human giant cell assays. This may reflect readier access of 5-LO metabolites to target cells in the isolated cell culture system.
Thus, the mechanisms by which leukotrienes and other systemic factors and hormones are responsible for osteoclast activation remain unknown. The connection between leukotrienes and other systemic hormones and bone resorbing factors and whether their effects on osteoclasts are related or independent was also unknown prior to the information described by the present application.
Inhibitors of 5-Lipoxygenase used herein include:
NGDA—nordihydroguaiaretic acid (see structure in FIG.
9
);
MK886—(see structure in FIG.
10
); and
ZM230,487—(see structure in
FIG. 11
)
SUMMARY OF THE INVENTION
The present invention focuses upon a method for inhibiting bone resorption. This method involves administering a 5-lipoxygenase inhibitor to a subject in an amount suppressing production of an osteoclast-stimulating factor. When the production of the osteoclast-stimulating factors via the 5-lipoxygenase pathway is inhibited, bone resorption markedly declines. The osteoclast-stimulating factors include peptido-leukotriene and 5-hydroxyeicosatetraenoic acid. Other factors yet to be identified or previously known may also be 5-lipoxygenase metabolites that stimulate bone resorption. While 5-lipoxygenase inhibitors may be substrate analogs or allosteric inhibitors, a substance which inhibits the activity of this enzyme may utilize other mechanisms (e.g., inhibition of 5-LO biosynthesis) and nevertheless function to inhibit bone resorption. Preferred inhibitors included NGDA, MK886 and ZM230,487. The best inhibitor thus far noted is ZM230,487.
The inhibition of bone resorption is highly desirable with, for example, periodontal disease, osteoporosis, estrogen deficiency, Paget's disease, inflammatory bone loss, bone malignancy, hyperparathyroidism and bone transplants. Administering of a 5-lipoxygenase inhibitor may be enteral—when oral administration is desired, parenteral, when appropriate (preferably by vascular injection or infusion), or topical such as application to oral tissues to prevent bone loss due to periodontal disease. T

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