BONE DELIVERY CONJUGATES AND METHOD OF USING SAME TO TARGET PROTEINS TO BONE
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 - 11 . (canceled)
12 . A bone delivery conjugate comprising sALP-Y-D n , wherein
Y is an amino acid sequence of at least one amino acid;
D n is a poly aspartate wherein n=10; and
sALP is a secreted soluble tissue non-specific alkaline phosphatase of SEQ ID NO: 6;
wherein said bone delivery conjugate is catalytically competent to allow formation of hydroxyapatite crystals in bone.
13 . The bone delivery conjugate of claim 12 , wherein said sALP is capable of catalyzing the cleavage of inorganic pyrophosphate (PPi).
14 . The bone delivery conjugate of claim 13 , 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 complementary to at least one of SEQ ID NO: 5 and SEQ ID NO: 7.
15 . The bone delivery conjugate of claim 14 , 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.
16 . A composition comprising the bone delivery conjugate of claim 12 and at least one pharmaceutically acceptable carrier.
17 . The composition of claim 16 , wherein said sALP is capable of catalyzing the cleavage of inorganic pyrophosphate (PPi).
18 . A method of delivering a protein to bone tissue of a mammal to treat a bone related undesired condition in said mammal, comprising administering to said mammal an effective amount of a bone delivery conjugate comprising sALP-Y-D n , wherein
Y is an amino acid sequence of at least one amino acid;
D n is a poly aspartate wherein n=10;
sALP is a secreted soluble tissue non-specific alkaline phosphatase of SEQ ID NO: 6; and
said bone delivery conjugate is catalytically competent to allow formation of hydroxyapatite crystals in bone.
19 . The method of claim 18 ,
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 complementary at least one of SEQ ID NO: 5 and 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.
20 . The method of claim 18 , wherein said mammal is a human, and the bone delivery conjugate further comprises at least one pharmaceutically acceptable carrier.
21 . The method of claim 20 , wherein said effective amount is administered to said human more than once at an interval ranging from daily to weekly.
22 . The method of claim 21 , wherein said effective amount is from about 1 to about 50 milligrams per kilogram of body weight.
23 . The method of claim 22 , wherein said effective amount is from about 1 to about 10 milligrams per kilogram of body weight.
24 . The method of claim 23 , wherein said effective amount is about 1 milligram per kilogram of body weight.
25 . The method of claim 20 , wherein said bone related undesired condition is at least one of hypophosphatasia (HPP), hypophosphatemic rickets, osteomalacia, a lack or insufficient amount of functional alkaline phosphatase, impaired skeletal mineralization, growth retardation, lower extremity deformity, hyperparathyroidism, parathyroidectomy, hypercalciuria, hypercalcemia, and nephrocalcinosis.
26 . The method of claim 25 , wherein the HPP is perinatal HPP, infantile HPP, childhood HPP, or adult HPP.
27 . The method of claim 20 , wherein the pharmaceutically acceptable carrier is saline.
28 . The method of claim 18 , wherein said sALP is capable of catalyzing the cleavage of inorganic pyrophosphate (PPi).
29 . The composition of claim 16 , wherein the pharmaceutically acceptable carrier is saline.