Bone delivery conjugates and method of using same to target proteins to bone
View Patent ↗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.
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.
31. The method of claim 30 , wherein Y is an amino acid sequence of at least one amino acid and X and Z are absent.
32. The method of claim 31 , wherein n=10.
33. The method of claim 30 , wherein said sALP is a secreted soluble form of said alkaline phosphatase.
34. The method of claim 30 , 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.
35. The method of claim 34 , wherein Y is an amino acid sequence of at least one amino acid and X and Z are absent, and wherein n=10.
36. The method of claim 19 , wherein said sALP is capable of catalyzing the cleavage of inorganic pyrophosphate (PPi).
37. The conjugate of claim 1 , wherein said composition is formulated for intravenous injection.
38. The composition of claim 9 , wherein said composition is formulated for parenteral administration.
39. The composition of claim 9 , wherein said composition is formulated for intravenous injection.
40. The method of claim 13 , wherein said conjugate is formulated for intravenous injection.
41. The method of claim 19 , wherein said conjugate is formulated for parenteral administration.
42. The method of claim 19 , wherein said conjugate is formulated for intravenous injection.
43. The conjugate of claim 1 , wherein said sALP is a human tissue non-specific alkaline phosphatase.
44. The conjugate of claim 1 , wherein Y lacks a transamination site.
45. The composition of claim 9 , wherein said sALP is a human tissue non-specific alkaline phosphatase.
46. The composition of claim 9 , wherein Y lacks a transamination site.
47. The composition of claim 9 , wherein said composition is formulated to provide 0.001 milligrams (mg) to 500 mg per kilogram (kg) of body weight per day of said conjugate.
48. The method of claim 13 , wherein said sALP is a human tissue non-specific alkaline phosphatase.
49. The method of claim 19 , wherein said sALP is a human tissue non-specific alkaline phosphatase.