Compositions and Methods for Modulating Osteoblast Cell Differentiation and Bone Generation Through HIF-1a

• US 20100015164A1
• Filed: 12/11/2006
• Published: 01/21/2010
• Est. Priority Date: 12/09/2005
• Status: Abandoned Application

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: UAB Research Foundation (University of Alabama System)
• Inventors: Clemens, Thomas L., Johnson, Randall S., Schipani, Ernestina
• Application Number:
  US12/096,733
• Publication Number:
  US 20100015164A1
• Time in Patent Office: Days
• US Class Current: 424/178.100
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/035 Animal model for multifactorial diseases
• CPC Class Codes: A01K 2267/0375 Animal model for cardiovascular diseases
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: G01N 2333/4706 stimulating, promoting or activating activity
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 2800/10 Musculoskeletal or connective tissue disorders
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere

Abstract

The present invention provides a novel screening tool for the determination of compounds capable of promoting bone healing, promoting osteoblast cellular differentiation, improving bone mass or volume, and/or promoting osteogenesis that could be used in the treatment of various bone-loss or bone density decreasing disorders. The present invention also provides a screening tool for the determination of compounds capable of inhibiting osteoblast cellular differentiation, decreasing bone mass or volume, and/or inhibiting osteogenesis. Also provided are compositions and methods for the treatment of various bone-loss or bone density decreasing disorders.

Claims

1. A method of identifying a compound that improves bone mass or volume, wherein the method comprises the steps of:

a. providing an osteoblast or bone precursor cell;

b. contacting the cell with a test compound; and

c. determining whether an increase in dimerization, nuclear translocation, and/or transcriptional activity of a HIF-1α

/HIF-1β

complex occurs in the cell contacted with the compound, said increase being an indication that the compound improves bone mass or volume.

2. The method of claim 1, wherein the transcriptional activity of a HIF-1α

/HIF-1β

complex is determined by measuring the transcription activation of a HIF-1α

target gene selected from the group consisting of VEGF;

nitric oxide synthase 2;

heme oxygenase 2;

α

_1B-adrenergic receptor;

erythropoietin;

transferrin;

ceruloplasmin;

transferrin receptor;

cyclin G2;

p21;

IGF-2;

IGF-binding protein 1, 2 and 3;

glucose transporter 1, and 3;

hexokinase 1, and 2;

phosphofructokinase L;

pyruvate kinase M;

a HIF-prolyl hydroxylase; and

carbonic anhydrase.

3. The method of claim 1, wherein the transcriptional activity of the HIF-1α

/HIF-1β

complex is determined by measuring the transcription activation of VEGF or Glut-1.

4. The method of claim 2, wherein the transcription activation is measured by real-time PCR.

5. The method of claim 1, wherein the expression level of HIF-1α

protein is measured.

6. The method of claim 5, wherein the expression level is measured by Western blotting.

7. The method of claim 1, wherein the method comprises an additional step:

d. determining whether an increase or decrease in collagen synthesis occurs in the cell contacted with the compound.

8. The method of claim 7, wherein the increase or decrease in collagen synthesis is detected by measuring the level of secreted collagen in the media.

9. The method of claim 8, wherein the level of secreted collagen is measured by Western blotting.

10. A process for making a compound that increases osteoblast cell differentiation, improves bone mass or volume, and/or promotes osteogenesis, comprising the steps of:

a. providing an osteoblast or bone precursor cell;

b. contacting the cell with a test compound;

c. determining whether an increase in dimerization, nuclear translocation, and/or transcriptional activity of a HIF-1α

/HIF-1β

complex occurs in the cell contacted with the compound, said increase being an indication that the compound increases osteoblasts cell differentiation, improves bone mass or volume, and/or promotes osteogenesis; and

d. manufacturing the compound.

11. A method of identifying a compound that promotes osteogenesis, wherein the method comprises the comprises the steps of:

a. providing an osteoblast or bone precursor cell;

b. contacting the cell with a test compound; and

c. determining whether an increase in dimerization, nuclear translocation, and/or transcriptional activity of a HIF-1α

/HIF-1β

complex occurs in the cell contacted with the compound, said increase being an indication that the compound promotes osteogenesis.

12. The method of claim 11, wherein the compound promotes osteogenesis by one or more of increasing osteoblast formation, increasing osteoid volume, decreasing osteoclast formation, and decreasing osteoclast function.

13. The method of claim 11, wherein the transcriptional activity of a HIF-1α

/HIF-1β

complex is determined by measuring the transcription activation of a HIF-1α

target gene selected from the group consisting of VEGF;

nitric oxide synthase 2;

heme oxygenase 2;

α

_1B-adrenergic receptor;

erythropoietin;

transferrin;

ceruloplasmin;

transferrin receptor;

cyclin G2;

p21;

IGF-2;

IGF-binding protein 1, 2 and 3;

glucose transporter I (Glut-1), and 3;

hexokinase 1, and 2;

phosphofructokinase L;

pyruvate kinase M;

a HIF-prolyl hydroxylase; and

carbonic anhydrase.

14. The method of claim 11, wherein the transcriptional activity of a HIF-1α

/HIF-1β

complex is determined by measuring the transcription activation of VEGF or Glut-1.

15. The method of claim 14, wherein the transcription activation is measured by real-time PCR.

16. The method of claim 11, wherein the expression level of HIF-1α

protein is measured.

17. The method of claim 16, wherein the expression is measured by Western blotting.

18. The method of claim 11, wherein the method comprises an additional step:

d. determining whether an increase or decrease in collagen synthesis occurs in the cell contacted with the compound.

19. The method of claim 18, wherein the increase or decrease in collagen synthesis is detected by measuring the level of secreted collagen in the media.

20. The method of claim 19, wherein the level of secreted collagen is measured by Western blotting.

21. A method of identifying a compound that increases osteoblast cell differentiation, wherein the method comprises the steps of:

a. providing an osteoblast or bone precursor cell;

b. contacting the cell with a test compound; and

c. determining whether an increase in dimerization, nuclear translocation, and/or transcriptional activity of an HIF-1α

/HIF-1β

complex occurs in the cell contacted with the compound, said increase being an indication that the compound increases osteoblast cell differentiation.

22. The method of claim 21, wherein the transcriptional activity of a HIF-1α

/HIF-1β

complex is determined by measuring the transcription activation of a HIF-1α

target gene selected from the group consisting of VEGF;

nitric oxide synthase 2;

heme oxygenase 2;

α

_1B-adrenergic receptor;

erythropoietin;

transferrin;

ceruloplasmin;

transferrin receptor;

cyclin G2;

p21;

IGF-2;

IGF-binding protein 3, 2 and 3;

glucose transporter 1, and 3;

hexokinase 1, and 2;

phosphofructokinase L;

pyruvate kinase M;

a HIF-prolyl hydroxylase; and

carbonic anhydrase.

23. The method of claim 21, wherein the transcriptional activity of tie HIF-1α

/HIF-1β

complex is determined by measuring the transcription activation of VEGF or Glut-1.

24. The method of claim 22, wherein the transcription activation is measured by real-time PCR.

25. - The method of claim 21, wherein the expression level of HIF-1α

protein is measured.

26. The method of claim 25, wherein the expression level is measured by Western blotting.

27. The method of claim 21, wherein the method comprises an additional step;

d. determining whether an increase or decrease in collagen synthesis occurs in the cell contacted with the compound.

28. The method of claim 27, wherein the increase or decrease in collagen synthesis is detected by measuring the level of secreted collagen in the media.

29. The method of claim 28, wherein the level of secreted collagen is measured by Western blotting.

30. The present invention also provides for methods of identifying a compound capable of decreasing osteoblast cell differentiation, decreasing bone mass or volume, and/or increasing osteoclast cell differentiation, wherein the methods

a. providing an osteoblast or bone precursor cell;

b. contacting the cell with a test compound; and

c. determining whether a decrease in dimerization, nuclear translocation, and/or transcriptional activity of a HIF-1α

/HIF-1β

complex occurs in the cell contacted with the compound, said decrease being an indication that the compound decreases osteoblast cell differentiation, decreases bone mass or volume, and/or increases osteoclast cell differentiation.

31. A process for making a compound that decreases osteoblast cell differentiation, decreases bone mass or volume, and/or increases osteoclast cell differentiation, comprising the steps of:

a. providing an osteoblast or bone precursor cell;

b. contacting the cell with a test compound; and

c. determining whether a decrease in dimerization, nuclear translocation, and/or transcriptional activity of a HIF-1α

/HIF-1β

complex occurs in the cell contacted with the compound, said decrease being an indication that the compound decreases osteoblast cell differentiation, decreases bone mass or volume, and/or increases osteoclast cell differentiation; and

d. manufacturing the compound.

32. A compound for use in promoting the activity of HIF-1α

for the treatment of a disease state, such as, but not limited to osteoporosis, osteopenia, or a bone-related disorder selected from the group consisting of osteoporosis, bone fractures, hypercalcemia of malignancy, osteopenia or osteolytic lesions due to bone metastases, periprosthetic osteolysis, familial expansile osteolysis, periodontal disease, tooth loss, rheumatoid arthritis, osteoarthritis, hyperparathyroidism, Paget'"'"'s disease, osteodystrophy, myositis ossificans, Bechterew'"'"'s disease, malignant hypercalcernia, bone loss, bone abnormalities due to steroid hormone treatment, bone abnormalities caused by cancer therapeutics, abnormally increased bone turnover, osteomalacia, Bechet'"'"'s disease, hyperostosis, osteopetrosis, osteogenesis imperfecta, rachitis, immobilization-induced osteopenia, expansile skeletal hyperphosphatasia, and glucocorticoid-induced osteoporosis.

33. A method for strengthening a bone graft, inducing vertebral synostosis, enhancing long bone extension, the treatment and promotion of healing of bone fractures and osteotomies, enhancing bone healing following facial reconstruction, maxillary reconstruction, and/or mandibular reconstruction in a vertebrate, comprising tie step of:

a. administering to said vertebrate a composition comprising a therapeutically effective amount of a compound of claim 32, a prodrug, or a pharmaceutically acceptable salt thereof, or a stereoisomer or diastereomeric mixture of said compound, prodrug, or salt.

34. The method of claim 33, wherein the composition is administered locally to the site of bone reconstruction.

35. The method of claim 33, wherein the composition is administered systemically.