Bone delivery conjugates and method of using same to target proteins to bone

Patent metadata

Field	Value
Publication number	US-10000532-B2
Application number	US-63852709-A
Country	US
Kind code	B2
Filing date	Dec 15, 2009
Priority date	Apr 21, 2004
Publication date	Jun 19, 2018
Grant date	Jun 19, 2018

Abstract

A bone delivery conjugate having a structure selected from the group consisting of: A) X-D n -Y-protein-Z; and B) Z-protein-Y-D n -X, wherein X is absent or is an amino acid sequence of at least one amino acid; Y is absent or is an amino acid sequence of at least one amino acid; Z is absent or is an amino acid sequence of at least one amino acid; and D n is a poly aspartate wherein n=10 to 16. Compositions comprising same and methods of use thereof.

First claim

What is claimed is:

1. A bone delivery conjugate comprising a structure selected from the group consisting of:
A) X-D n -Y-sALP-Z; and
B) Z-sALP-Y-D n -X,
wherein X is absent or is an amino acid sequence of at least one amino acid;
Y is absent or is an amino acid sequence of at least one amino acid;
Z is absent or is an amino acid sequence of at least one amino acid;
D n is a poly aspartate wherein n=10 to 16;
said sALP is a soluble tissue non-specific alkaline phosphatase; and
said bone delivery conjugate is catalytically competent to improve skeletal mineralization in bone.

2. The bone delivery conjugate of claim 1 , wherein said structure is: Z-sALP-Y-D n -X.

3. The bone delivery conjugate of claim 2 , wherein Y is an amino acid sequence of at least one amino acid and X and Z are absent.

4. The bone delivery conjugate of claim 3 , wherein n=10.

5. The bone delivery conjugate of claim 2 , wherein said sALP is a secreted soluble form of said alkaline phosphatase.

6. The bone delivery conjugate of claim 2 , wherein said bone delivery conjugate is encoded by a nucleic acid molecule which hybridizes under high stringency conditions to a nucleic acid molecule having a nucleic acid sequence completely complementary to SEQ ID NO: 7.

7. The bone delivery conjugate of claim 6 , wherein said high stringency conditions comprise pre-hybridization and hybridization in 6×SSC, 5×Denhardt's reagent, 0.5% SDS and 100 mg/ml of denatured fragmented salmon sperm DNA at 68° C.; and washes in 2×SSC and 0.5% SDS at room temperature for 10 minutes; in 2×SSC and 0.1% SDS at room temperature for 10 minutes; and in 0.1×SSC and 0.5% SDS at 65° C. three times for five minutes.

8. The bone delivery conjugate of claim 7 , wherein Y is an amino acid sequence of at least one amino acid and X and Z are absent, and wherein n=10.

9. A bone delivery composition comprising a pharmaceutically acceptable carrier and a bone delivery conjugate comprising a structure selected from the group consisting of:
A) X-D n -Y-sALP-Z; and
B) Z-sALP-Y-D n -X,
wherein X is absent or is an amino acid sequence of at least one amino acid;
Y is absent or is an amino acid sequence of at least one amino acid;
Z is absent or is an amino acid sequence of at least one amino acid;
D n is a poly aspartate wherein n=10 to 16;
said sALP is a soluble tissue non-specific alkaline phosphatase; and
said bone delivery conjugate is catalytically competent to improve skeletal mineralization in bone.

10. The bone delivery composition of claim 9 , wherein said structure is: Z-sALP-Y-D n -X.

11. The bone delivery composition of claim 10 , wherein said bone delivery conjugate is encoded by a nucleic acid molecule which hybridizes under high stringency conditions to a nucleic acid molecule having a nucleic acid sequence completely complementary to SEQ ID NO: 7, wherein said high stringency conditions comprise pre-hybridization and hybridization in 6×SSC, 5×Denhardt's reagent, 0.5% SDS and 100 mg/ml of denatured fragmented salmon sperm DNA at 68° C.; and washes in 2×SSC and 0.5% SDS at room temperature for 10 minutes; in 2×SSC and 0.1% SDS at room temperature for 10 minutes; and in 0.1×SSC and 0.5% SDS at 65° C. three times for five minutes.

12. The bone delivery composition of claim 11 , wherein Y is an amino acid sequence of at least one amino acid and X and Z are absent, and wherein n=10.

13. A method of delivering a protein to bone tissue of a mammal comprising administering to said mammal an effective amount of a bone delivery conjugate comprising a structure selected from the group consisting of:
A) X-D n -Y-sALP-Z; and
B) Z-sALP-Y-D n -X,
wherein X is absent or is an amino acid sequence of at least one amino acid;
Y is absent or is an amino acid sequence of at least one amino acid;
Z is absent or is an amino acid sequence of at least one amino acid;
D n is a poly aspartate wherein n=10 to 16;
said sALP is a soluble tissue non-specific alkaline phosphatase; and
said bone delivery conjugate is catalytically competent to improve skeletal mineralization in bone.

14. The method of claim 13 ,
wherein said structure is: Z-sALP-Y-D n -X;
said bone delivery conjugate is encoded by a nucleic acid molecule which hybridizes under high stringency conditions to a nucleic acid molecule having a nucleic acid sequence completely complementary to SEQ ID NO: 7;
said high stringency conditions comprise pre-hybridization and hybridization in 6×SSC, 5×Denhardt's reagent, 0.5% SDS and 100 mg/ml of denatured fragmented salmon sperm DNA at 68° C.; and washes in 2×SSC and 0.5% SDS at room temperature for 10 minutes; in 2×SSC and 0.1% SDS at room temperature for 10 minutes; and in 0.1×SSC and 0.5% SDS at 65° C. three times for five minutes;
Y is an amino acid sequence of at least one amino acid and X and Z are absent; and n=10.

15. The method of claim 13 , wherein said mammal is human.

16. The method of claim 15 , wherein said effective amount is 1 to 10 milligrams per kilogram of body weight.

17. The method of claim 16 , wherein said effective amount is administered to said human more than once at an interval ranging from daily to weekly.

18. The method of claim 15 , wherein said effective amount is about 1 milligram per kilogram of body weight.

19. A method of treating a condition or disease related to a bone defect characterized by a lack of or an insufficient amount of functional alkaline phosphatase comprising administering to a mammal in need thereof an effective amount of a bone delivery conjugate comprising a structure selected from the group consisting of:
A) X-D-Y-sALP-Z; and
B) Z-sALP-Y-D-X,
wherein X is absent or is an amino acid sequence of at least one amino acid;
Y is absent or is an amino acid sequence of at least one amino acid;
Z is absent or is an amino acid sequence of at least one amino acid;
D n is a poly aspartate wherein n=10 to 16;
said sALP is a soluble tissue non-specific alkaline phosphatase; and
said bone delivery conjugate is catalytically competent to improve skeletal mineralization in bone,
wherein the conjugate is in a pharmaceutically acceptable carrier.

20. The method of claim 19 , wherein said condition or disease is hypophosphatasia.

21. The method of claim 19 , wherein said mammal is human.

22. The method of claim 21 , wherein said effective amount is 1 to 10 milligrams per kilogram of body weight.

23. The method of claim 22 , wherein said effective amount is administered to said human more than once at an interval ranging from daily to weekly.

24. The method of claim 21 , wherein said effective amount is about 1 milligram per kilogram of body weight.

25. The bone delivery conjugate of claim 1 , wherein said sALP is capable of catalyzing the cleavage of inorganic pyrophosphate (PPi).

26. The bone delivery composition of claim 10 , wherein Y is an amino acid sequence of at least one amino acid and X and Z are absent.

27. The bone delivery composition of claim 26 , wherein n=10.

28. The bone delivery composition of claim 10 , wherein said sALP is a secreted soluble form of said alkaline phosphatase.

29. The bone delivery composition of claim 9 , wherein said sALP is capable of catalyzing the cleavage of inorganic pyrophosphate (PPi).

30. The method of claim 19 , wherein said structure is: Z-sALP-Y-D n -X.

Assignees

• Crine Philippe
• Boileau Guy
• Lemire Isabelle
• Loisel Thomas P
• Alexion Pharma Inc

Inventors

• Crine Philippe
• Boileau Guy
• Lemire Isabelle
• Loisel Thomas P

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P19/00

  Drugs for skeletal disorders · CPC title

• A61P19/08

  for bone diseases, e.g. rachitism, Paget's disease · CPC title

• C07K7/06 Primary

  having 5 to 11 amino acids · CPC title

• C12N9/6494

  Neprilysin (3.4.24.11), i.e. enkephalinase or neutral-endopeptidase 24.11 · CPC title