Heparin and heparan sulfate oligosaccharides
阅读说明：本技术 肝素和硫酸乙酰肝素寡糖 (Heparin and heparan sulfate oligosaccharides ) 是由 R·A·A·史密斯 S·M·库尔 V·纳康比 于 2018-12-11 设计创作，主要内容包括：公开了能够结合BMP2的链长为至少10个糖且不超过50个糖的分离的肝素或硫酸乙酰肝素寡糖片段。还公开了相同的肝素或硫酸乙酰肝素寡糖片段在试剂盒和药物组合物中的用途,以及相同的硫酸乙酰肝素寡糖片段在结缔组织和骨骼的修复和/或再生以及伤口治疗中的用途。(Isolated heparin or heparan sulfate oligosaccharide fragments having a chain length of at least 10 sugars and no more than 50 sugars capable of binding BMP2 are disclosed. Also disclosed are uses of the same heparin or heparan sulfate oligosaccharide fragments in kits and pharmaceutical compositions, and uses of the same heparan sulfate oligosaccharide fragments in the repair and/or regeneration of connective tissue and bone and in wound therapy.)
1. An isolated heparin or heparan sulfate oligosaccharide having a chain length of at least 10 saccharides and no more than 50 saccharides, wherein the oligosaccharide is capable of binding BMP 2.
2. The isolated heparin or heparan sulfate oligosaccharide of claim 1, wherein said isolated heparin or heparan sulfate has a chain length of at least 12 sugars.
3. The isolated heparin or heparan sulfate oligosaccharide of claim 1, wherein said isolated heparin or heparan sulfate comprises or consists of a chain length of about 10 or about 12 sugars.
4. The isolated heparin or heparan sulfate oligosaccharide of claim 1, wherein the isolated heparin or heparan sulfate comprises or consists of a chain length of 10 to 50 sugars, optionally one of 12 to 50 sugars, 14 to 50 sugars, 16 to 50 sugars, 18 to 50 sugars, 20 to 50 sugars, 22 to 50 sugars, 24 to 50 sugars, 26 to 50 sugars, 28 to 50 sugars, 30 to 50 sugars, 32 to 50 sugars, 34 to 50 sugars, 36 to 50 sugars, 38 to 50 sugars, 40 to 50 sugars, 42 to 50 sugars, 44 to 50 sugars, 46 to 50 sugars, or 48 to 50 sugars.
5. The isolated heparin or heparan sulfate oligosaccharide of claim 1, wherein the isolated heparin or heparan sulfate comprises or consists of a chain length of 10 to 36 sugars, optionally one of 12 to 36 sugars, 14 to 36 sugars, 16 to 36 sugars, 18 to 36 sugars, 20 to 36 sugars, 22 to 36 sugars, 24 to 36 sugars, 26 to 36 sugars, 28 to 36 sugars, 30 to 36 sugars, 32 to 36 sugars, or 34 to 36 sugars.
6. The isolated heparin or heparan sulfate oligosaccharide of claim 1, wherein the isolated heparin or heparan sulfate comprises or consists of a chain length of 36 to 50 sugars, optionally one of 38 to 50 sugars, 40 to 50 sugars, 42 to 50 sugars, 44 to 50 sugars, 46 to 50 sugars, or 48 to 50 sugars.
7. The isolated heparin or heparan sulfate oligosaccharide of claim 1, wherein the isolated heparin or heparan sulfate comprises or consists of a chain length of 18 to 40 sugars, optionally one of 20 to 40 sugars, 22 to 40 sugars, 24 to 40 sugars, 26 to 40 sugars, 28 to 40 sugars, 30 to 40 sugars, 32 to 40 sugars, 34 to 40 sugars, 36 to 40 sugars, or 38 to 40 sugars.
8. The isolated heparin or heparan sulfate oligosaccharide according to any one of claims 1 to 7, wherein said isolated heparin or heparan sulfate enhances BMP 2-mediated ALP activity, as measured in an in vitro assay of BMP 2-mediated ALP activity.
9. The isolated heparin or heparan sulfate oligosaccharide of any one of claims 1 to 8, wherein the isolated heparin or heparan sulfate enhances BMP 2-mediated Smad1/5/9 phosphorylation, e.g., as measured in an in vitro assay of BMP 2-mediated Smad1/5/9 phosphorylation.
10. The isolated heparin or heparan sulfate oligosaccharide of any one of claims 1 to 9, wherein said isolated heparin or heparan sulfate is N-sulfated.
11. The isolated heparin or heparan sulfate oligosaccharide of any one of claims 1 to 10, wherein said isolated heparin or heparan sulfate is 6-O sulfated.
12. The isolated heparin or heparan sulfate oligosaccharide of any one of claims 1 to 11, wherein said isolated heparin or heparan sulfate is 2-O desulphated.
13. The isolated heparin or heparan sulfate oligosaccharide mixture according to any one of claims 1 to 12.
14. A mixture comprising or consisting of a plurality of heparin or heparan sulfate oligosaccharides that bind BMP2, each of said heparin or heparan sulfate oligosaccharides having a chain length of at least 10 sugars and no more than 50 sugars.
15. The mixture of claim 14, wherein the mixture is provided in an isolated or substantially pure form.
16. The mixture of claim 14 or 15, wherein the oligosaccharides each have a chain length of at least 12 sugars.
17. The mixture of claim 14 or 15, wherein the oligosaccharides each have a chain length of about 10 or about 12 sugars.
18. The mixture of any one of claims 14 to 17, wherein the oligosaccharides each have a chain length of 10 to 50 sugars, optionally one of 12 to 50 sugars, 14 to 50 sugars, 16 to 50 sugars, 18 to 50 sugars, 20 to 50 sugars, 22 to 50 sugars, 24 to 50 sugars, 26 to 50 sugars, 28 to 50 sugars, 30 to 50 sugars, 32 to 50 sugars, 34 to 50 sugars, 36 to 50 sugars, 38 to 50 sugars, 40 to 50 sugars, 42 to 50 sugars, 44 to 50 sugars, 46 to 50 sugars, or 48 to 50 sugars.
19. The mixture of any one of claims 14 to 17, wherein the oligosaccharides each have a chain length of 10 to 36 saccharides, optionally one of 12 to 36 saccharides, 14 to 36 saccharides, 16 to 36 saccharides, 18 to 36 saccharides, 20 to 36 saccharides, 22 to 36 saccharides, 24 to 36 saccharides, 26 to 36 saccharides, 28 to 36 saccharides, 30 to 36 saccharides, 32 to 36 saccharides, or 34 to 36 saccharides.
20. The mixture of any one of claims 14 to 17, wherein the oligosaccharides each have a chain length of 36 to 50 sugars, optionally one of 38 to 50 sugars, 40 to 50 sugars, 42 to 50 sugars, 44 to 50 sugars, 46 to 50 sugars, or 48 to 50 sugars.
21. The mixture of any one of claims 14 to 17, wherein the oligosaccharides each have a chain length of 18 to 40 sugars, optionally one of 20 to 40 sugars, 22 to 40 sugars, 24 to 40 sugars, 26 to 40 sugars, 28 to 40 sugars, 30 to 40 sugars, 32 to 40 sugars, 34 to 40 sugars, 36 to 40 sugars, or 38 to 40 sugars.
22. The mixture of any of claims 14 to 21, wherein the heparin or heparan sulfate oligosaccharide that binds BMP2 enhances BMP 2-mediated ALP activity, e.g., as measured in an in vitro assay of BMP 2-mediated ALP activity.
23. The mixture of any one of claims 14 to 22, wherein the heparin or heparan sulfate oligosaccharide that binds BMP2 enhances BMP 2-mediated Smad1/5/9 phosphorylation, for example as measured in an in vitro assay of BMP 2-mediated Smad1/5/9 phosphorylation.
24. The mixture according to any one of claims 14 to 23, wherein the heparin or heparan sulfate oligosaccharide that binds BMP2 is N-sulfated.
25. The mixture according to any one of claims 14 to 24, wherein the heparin or heparan sulfate oligosaccharide binding to BMP2 is 6-O sulfated.
26. The mixture according to any one of claims 14 to 25, wherein the heparin or heparan sulfate oligosaccharide binding to BMP2 is 2-O desulphated.
27. Optionally an isolated fragment of heparan sulphate that binds BMP2, wherein the fragment comprises or consists of an oligosaccharide with a chain length of at least 10 saccharides, wherein the oligosaccharide is capable of binding BMP 2.
28. The fragment of claim 27, wherein said heparan sulfate that binds BMP2 is HS 3.
29. A fragment according to claim 27 or 28, wherein the oligosaccharide has a chain length of no more than 50 saccharides.
30. The fragment of any one of claims 27 to 29, wherein said oligosaccharide has a chain length of at least 12 sugars.
31. The fragment of any one of claims 27 to 30, wherein said oligosaccharide has a chain length of about 10 or about 12 sugars.
32. The fragment of any one of claims 27 to 31, wherein said oligosaccharide comprises or consists of a chain length of 10 to 50 saccharides, optionally one of 12 to 50 saccharides, 14 to 50 saccharides, 16 to 50 saccharides, 18 to 50 saccharides, 20 to 50 saccharides, 22 to 50 saccharides, 24 to 50 saccharides, 26 to 50 saccharides, 28 to 50 saccharides, 30 to 50 saccharides, 32 to 50 saccharides, 34 to 50 saccharides, 36 to 50 saccharides, 38 to 50 saccharides, 40 to 50 saccharides, 42 to 50 saccharides, 44 to 50 saccharides, 46 to 50 saccharides, or 48 to 50 saccharides.
33. The fragment of any one of claims 27 to 31, wherein said oligosaccharide comprises or consists of a chain length of 10 to 36 saccharides, optionally one of 12 to 36 saccharides, 14 to 36 saccharides, 16 to 36 saccharides, 18 to 36 saccharides, 20 to 36 saccharides, 22 to 36 saccharides, 24 to 36 saccharides, 26 to 36 saccharides, 28 to 36 saccharides, 30 to 36 saccharides, 32 to 36 saccharides or 34 to 36 saccharides.
34. The fragment of any one of claims 27 to 31, wherein said oligosaccharide comprises or consists of a chain length of 36 to 50 saccharides, optionally one of 38 to 50 saccharides, 40 to 50 saccharides, 42 to 50 saccharides, 44 to 50 saccharides, 46 to 50 saccharides or 48 to 50 saccharides.
35. The fragment of any one of claims 27 to 31, wherein said oligosaccharide comprises or consists of a chain length of 18 to 40 saccharides, optionally one of 20 to 40 saccharides, 22 to 40 saccharides, 24 to 40 saccharides, 26 to 40 saccharides, 28 to 40 saccharides, 30 to 40 saccharides, 32 to 40 saccharides, 34 to 40 saccharides, 36 to 40 saccharides or 38 to 40 saccharides.
36. The fragment of any one of claims 27 to 35, wherein said fragment enhances BMP2 mediated ALP activity, e.g. as measured in an in vitro assay for BMP2 mediated ALP activity.
37. The fragment of any one of claims 27 to 36, wherein the fragment enhances BMP 2-mediated Smad1/5/9 phosphorylation, for example as measured in an in vitro assay for BMP 2-mediated Smad1/5/9 phosphorylation.
38. The fragment of any one of claims 27 to 37, wherein said heparin or heparan sulfate is N-sulfated.
39. The fragment of any one of claims 27 to 38, wherein said heparin or heparan sulfate is 6-O sulfated.
40. The fragment of any one of claims 27 to 39, wherein said heparin or heparan sulfate is 2-O desulphated.
41. A mixture comprising or consisting of a plurality of fragments of any one of claims 27 to 40.
42. A pharmaceutical composition or medicament comprising the isolated heparin or heparan sulfate of any one of claims 1 to 12, the mixture of claims 13 to 26 or 41, or the fragment of claims 27 to 40.
43. The isolated heparin or heparan sulfate according to any one of claims 1 to 12, the mixture according to claims 13 to 26 or 41 or the fragment according to claims 27 to 40 for use in a method of treatment.
44. Use of the isolated heparin or heparan sulfate according to any one of claims 1 to 12, the mixture according to claims 13 to 26 or 41 or the fragment according to claims 27 to 40 for the manufacture of a medicament for use in a method of treatment.
45. A method of treatment comprising the step of administering the isolated heparin or heparan sulfate of any one of claims 1 to 12, the mixture of claims 13 to 26 or 41, or the fragment of claims 27 to 40 to a subject in need of treatment.
46. The isolated heparin or heparan sulfate of claim 43 for use in a method of treatment, the use of claim 44 or the method of treatment of claim 45, wherein the method of treatment comprises:
a method of wound healing in vivo,
-the repair and/or regeneration of connective tissue,
-the repair and/or regeneration of bone,
-the repair and/or regeneration of bones in mammals or humans, or
-repair and/or regeneration of bone fractures.
47. A biocompatible implant or prosthesis comprising a biomaterial and the isolated heparin or heparan sulfate of any one of claims 1 to 12, the mixture of claims 13 to 26 or 41 or the fragment of claims 27 to 40.
48. A method of forming a biocompatible implant or prosthesis, the method comprising the step of coating or impregnating a biomaterial with the isolated heparin or heparan sulfate of any one of claims 1 to 12, the mixture of claims 13 to 26 or 41 or the fragments of claims 27 to 40.
49. A method of treating a bone fracture in a patient, the method comprising surgically implanting into the fracture site or tissue surrounding the fracture site of the patient a biocompatible implant or prosthesis comprising a biomaterial and the isolated heparin or heparan sulfate of any one of claims 1 to 12, the mixture of claims 13 to 26 or 41 or the fragment of claims 27 to 40.
50. A kit of parts comprising a predetermined amount of the isolated heparin or heparan sulfate according to any one of claims 1 to 12, the mixture according to claims 13 to 26 or 41 or the fragment according to claims 27 to 40, and a predetermined amount of BMP 2.
51. A product comprising a therapeutically effective amount of:
(i) the isolated heparin or heparan sulfate of any one of claims 1 to 12, the mixture of claims 13 to 26 or 41, or the fragment of claims 27 to 40; and
(ii) BMP2 protein;
for simultaneous, separate or sequential use in a method of treatment.
Technical Field
The present invention relates to heparin and heparan sulphate oligosaccharides, including heparin-type or heparan sulphate-type oligosaccharides having a defined chain length, and in particular, but not exclusively, to such oligosaccharides that bind
Background
Bone Morphogenetic Proteins (BMPs) are a large group of transforming growth factor-beta (TGF- β) superfamily, which play a crucial role in a variety of processes including mesoderm formation, neural patterns, skeletal development and limb formation (1, 2). More than 15 BMP members have been identified, which play an important role in the processes of tissue repair and remodeling following injury (3-7). In animal models, several recombinant BMPs have been reported to induce ectopic bone formation and enhance healing of critical-size segmental bone defects. Clinical studies have shown that the use of recombinant human BMP is a safe and effective alternative to autologous bone grafting. Recombinant BMP2 and BMP-7 were approved for use in human spinal fusion and recalcitrant long bone nonunion, respectively (4-7).
At the cellular level, BMP signaling is initiated by binding to two types of specific transmembrane serine/threonine kinase receptors, type I (BMPR-I) and type II (BMPR-II) receptors (8). BMP2 signalling has been shown to be due to binding to the pre-aggregated receptor complex, rather than to free receptors in the cell membrane (9). Upon ligand binding, the type I receptor is activated by the ligand-bound type II receptor. The activated type I receptor then phosphorylates Smad1,
The biological activity of BMP in vivo and in vitro has been reported to be positively and negatively regulated by a number of extracellular and cell surface sulfated polysaccharides such as heparin and Heparan Sulfate (HS) (8, 13-20). BMP2 transformed the differentiation pathway of C2C12 myoblasts into osteoblast lineage, heparin enhanced BMP 2-induced osteoblast differentiation in C2C12 myoblasts in vitro (20-22). Like other proteins, it is believed that specific sizes and sulfated residues in the heparin/heparan sulfate chain bind to BMP2, thereby modulating receptor-mediated signaling of these molecules.
Over the years, hundreds of HS binding proteins have been identified, but how HS interacts with these proteins and affects their stability, concentration, conformation and activity is a fundamental issue in biology. Several studies have shown that binding of growth factors to HS and thus mitotic activity occurs only when specific structural features are present in the HS chain (23). These characteristics include sulfation of specific positions within the disaccharide; of particular importance are the 6-O-sulfated glucosamine-N-sulfate and the 2-O-sulfated iduronic acid residues, the minimum binding sequence usually being at least 5-6 disaccharides in length (24-26). The precise structure of the HS involved in these interactions remains elusive. Information on the minimal binding sequence on HS is crucial for understanding the rules of HS-protein interactions and the design of HS mimetics that can target proteins in human diseases. The minimal length and structural features of the heparin/HS motif were identified in only a few cases (27-30). An example of the first intensive study on the minimum length of the HS motif required for ligand binding and activity is that heparin-derived pentasaccharides are sufficient to interact with antithrombin III to inhibit coagulation factors thrombin (IIa) and coagulation factor Xa (28-30). However, heparin-derived tetrasaccharides are sufficient to interact with fibroblast growth factor 2(FGF-2), but oligosaccharides with a degree of polymerization (dp) of 10-12 are required to optimize the proliferative activity of FGF-2. However, the structural sequence required for antithrombin III binding is most characterized by an aberrant 3-O sulfate group on the 6-O sulfate N-sulfoglucosamine residue (31). Furthermore, the structural features of the HS pattern that can bind FGF-2 indicate that the continuous extension of the disulfide disaccharide N-sulfoglucosamine-iduronic acid 2-O sulfate is very important for obtaining the highest binding capacity (32). These examples clearly show that the minimum oligosaccharide length unit required for either binding or biological activity and the specific structural features of the HS pattern are not strictly related.
In this case, the characterization of the smallest HS unit and the specific sulfate group of HS binding to BMP2 is very important in an attempt to elucidate the molecular mechanisms that potentiate the regulatory requirements of HS at BMP2 activity. However, HS varies greatly in sequence and size and is available from a limited number of sources. Heparin has a structure similar to the sulfated regions of HS. Thus, in this study, we investigated the relationship between specific sulfate groups and the minimum length of heparin-derived oligosaccharides (which is necessary for binding to BMP2) and their ability to enhance BMP 2-induced osteoblast differentiation on C2C12 myoblasts.
Previously, we identified and isolated heparan sulfate that binds BMP2 with high affinity and demonstrated that this HS is active in the repair/regeneration of bone. Our work is reported in WO2010/030244a1 and US 9498494.
Summary of The Invention
In one aspect of the invention, there is provided an isolated heparin or heparan sulfate oligosaccharide having a chain length of at least 6 sugars and no more than 50 sugars.
Other aspects and embodiments are described in the appended claims.
In some embodiments, the chain length of the oligosaccharide is one of: at least 8, at least 10, or at least 12 saccharides.
In some preferred embodiments, the oligosaccharide comprises or consists of a chain length of 8, 10 or 12 saccharides.
In some embodiments, the oligosaccharide has a chain length of no more than one of: 49. 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 saccharides.
In some embodiments, the oligosaccharide has a chain length of one of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 sugars.
In some embodiments, the oligosaccharide is N-sulfated. This may include N-sulfation of the N-acetyl-D-glucosamine (GlcNAc) residue in the oligosaccharide chain of heparin or heparan sulfate. Preferably, at least 80% of the N-acetyl-D-glucosamine (GlcNAc) residues in the isolated heparin or heparan sulfate are N-sulfated. In some embodiments, it may be one of at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.
In some embodiments, the oligosaccharide is 6-O-sulfated (O-sulfation of an N-sulfoglucosamine (GlcNS) residue at C6). Preferably, at least 80% of the N-sulfoglucosamine (GlcNS) residues in the heparin or heparan sulfate oligosaccharide chains are 6-O-sulfated. In some embodiments, it may be one of at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.
Although oligosaccharides may be variously 2-O sulfated (O-sulfated at C2 of IdoA and GlcA), in certain embodiments, isolated heparin or heparan sulfate is 2-O-desulphated.
In some embodiments, the oligosaccharide binds BMP2 protein, optionally with a K less than one ofD: 100mM, 50mM, 10mM, 1mM, 100nM, 50nM, 10nM, or 1 nM.
Formulations or compositions comprising isolated heparin or heparan sulfate are provided.
In one aspect of the invention, there is provided a pharmaceutical composition or medicament comprising an isolated heparin or heparan sulfate as described in the above aspects. The pharmaceutical composition or medicament may further comprise a pharmaceutically acceptable carrier, adjuvant or diluent.
In another aspect of the invention, there is provided a composition comprising isolated heparin or heparan sulfate according to any one of the above aspects and BMP2 protein.
In one aspect of the invention, isolated heparin or heparan sulfate is provided for use in a method of treatment.
In a related aspect of the invention, there is provided the use of isolated heparin or heparan sulphate in the manufacture of a medicament for use in a method of treatment.
In another aspect of the invention, a method of treatment is provided, the method comprising the step of administering isolated heparin or heparan sulfate to a subject in need of treatment.
In aspects of the invention that relate to methods of treatment, the methods of treatment may include methods of wound healing in vivo, repair and/or regeneration of connective tissue, repair and/or regeneration of bone, and/or repair and/or regeneration of bone in a mammal or human. In some preferred embodiments, the method of treatment comprises repair and/or regeneration of a bone fracture. In some embodiments, the method of treatment may comprise the simultaneous or sequential administration of BMP2 protein.
In some embodiments, a method of treating a bone fracture in a patient is provided, the method comprising administering to the patient a therapeutically effective amount of isolated heparin or heparan sulfate. In some embodiments, the method comprises administering isolated heparin or heparan sulfate to tissue surrounding the fracture. In some embodiments, the method comprises injecting isolated heparin or heparan sulfate into the tissue surrounding the fracture. In such methods, the isolated heparin or heparan sulfate may be formulated into a pharmaceutical composition or medicament comprising isolated heparin or heparan sulfate and a pharmaceutically acceptable carrier, adjuvant or diluent.
In some embodiments, the method may further comprise administering BMP2 protein to the patient. In such methods, the isolated heparin or heparan sulfate and BMP2 protein may be formulated into a pharmaceutical composition comprising the isolated heparin or heparan sulfate and BMP2 protein, and a pharmaceutically acceptable carrier, adjuvant, or diluent.
In another aspect of the invention, a biocompatible implant or prosthesis is provided comprising a biomaterial and isolated heparin or heparan sulfate. In some embodiments, the implant or prosthesis is coated with isolated heparin or heparan sulfate. In some embodiments, the implant or prosthesis is impregnated with isolated heparin or heparan sulfate.
In another aspect of the invention, a method of forming a biocompatible implant or prosthesis is provided, the method comprising the step of coating or impregnating a biomaterial with isolated heparin or heparan sulfate. In some embodiments, the method further comprises coating or impregnating the biomaterial with BMP2 protein.
In some embodiments, a method of treating a bone fracture in a patient is provided, the method comprising surgically implanting into the tissue at or around the bone fracture site of the patient a biocompatible implant or prosthesis comprising a biomaterial and isolated heparin or heparan sulfate.
In some embodiments, the implant or prosthesis is coated with isolated heparin or heparan sulfate. In some embodiments, the implant or prosthesis is impregnated with isolated heparin or heparan sulfate. In some embodiments, the implant or prosthesis is further impregnated with BMP2 protein.
In another aspect of the invention, a kit of parts is provided, said kit comprising a predetermined amount of isolated heparin or heparan sulfate, and a predetermined amount of
In another aspect of the invention, there is provided a product comprising therapeutically effective amounts of:
(i) isolated heparin or heparan sulfate; and
(ii) BMP2 protein;
for simultaneous, separate or sequential use in a medical procedure. The medical treatment methods may include methods of wound healing in vivo, repair and/or regeneration of connective tissue, repair and/or regeneration of bone, and/or repair and/or regeneration of bone in a mammal or human. The products may optionally be formulated as a combined preparation for co-administration.
In another aspect of the invention there is provided a method of designing heparin or heparan sulphate, optionally heparin or heparan sulphate for use in a method of treatment according to the invention, the method comprising determining one or more of the chain length, sulphation pattern, and sugar (or disaccharide) content or sequence of heparin or heparan sulphate having BMP2 binding activity.
In another aspect of the invention, there is provided a method of making, producing or preparing heparin or heparan sulphate, optionally heparin or heparan sulphate for use in the treatment methods of the invention, the method comprising one or more of the following steps:
(i) determining one or more of the chain length, sulfation pattern, and saccharide (or disaccharide) content or sequence of heparin or heparan sulfate having BMP2 binding activity;
(ii) synthesizing one or more heparin or heparan sulfate oligosaccharides having a chain length, and/or sulfation pattern and/or sugar (or disaccharide) content or sequence associated with BMP2 binding activity;
(iii) one or more heparins or heparan sulphate oligosaccharides having a chain length, and/or sulphation pattern and/or sugar (or disaccharide) content or sequence associated with BMP2 binding activity are formulated as a pharmaceutical composition or medicament.
Detailed Description
In some embodiments, heparin or heparan sulfate oligosaccharides can be obtained by size fractionation of heparin or heparan sulfate preparations. Thus, the heparin and heparan sulfate oligosaccharides can be larger heparin or heparan sulfate molecular fragments. Suitable sources for size fractionated heparin and heparan sulfate preparations include commercially available heparin and heparan sulfate preparations. For example, a heparan sulfate preparation may be obtained during the separation of heparin from porcine intestinal mucosa (e.g. available from the kresol laboratory company of sigma aldrich, aldron, uk).
Other suitable sources of heparin or heparan sulfate include heparin or heparan sulfate from any mammal (human or non-human), particularly from the renal, pulmonary or intestinal mucosa. In some embodiments, the heparin or heparan sulfate is from porcine (porcine) or bovine (bovine) intestinal mucosa, kidney or lung.
In other embodiments, heparin and heparan sulfate oligosaccharides can be obtained by chemical synthesis of the desired oligosaccharide chains.
The heparin or heparan sulfate oligosaccharides or fragments of the invention may be provided in isolated form or in substantially purified form. This may include providing a composition wherein the heparin or heparan sulfate oligosaccharide component is at least 80% heparin or heparan sulfate oligosaccharide, more preferably at least one of 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.
Glycosaminoglycans
As used herein, the terms "glycosaminoglycan" and "GAG" are used interchangeably and are understood to refer to a large collection of molecules comprising oligosaccharides in which one or more of these binding sugars have an amino substituent or derivative thereof. Examples of GAGs are chondroitin sulfate, keratan sulfate, heparin, dermatan sulfate, hyaluronate and heparan sulfate.
Heparin
Heparin is a highly sulfated glycosaminoglycan. The most common disaccharide units in heparin are 2-O sulfated iduronic acid and 6-O sulfated, N-sulfated glucosamine, IdoA (2S) -GlcNS (6S). This accounts for approximately 85% of heparin in the beef lung and approximately 75% of heparin in the porcine intestinal mucosa. Although related to heparan sulfate, heparan sulfate differs from heparin in that heparan sulfate generally consists of glucuronic acid (GlcA) linked to N-acetylglucosamine (GlcNAc), accounting for about 50% of the total disaccharide units.
Heparan Sulfate (HS)
Heparan Sulfate Proteoglycans (HSPGs) represent a highly diverse subset of proteoglycans, consisting of heparan sulfate glycosaminoglycan side chains covalently linked to a protein backbone. Core protein exists in three major forms: a secreted form known as perlecan, a form known as glypican anchored in the plasma membrane, and a transmembrane form known as cohesin. They are ubiquitous components of mammalian cell surfaces and most extracellular matrices. Other proteins such as agrin or amyloid precursor protein exist in which HS chains can be attached to the less common nucleus.
"heparan sulfate" ("heparan sulfate" or "HS") is initially synthesized in the Golgi apparatus as a polysaccharide consisting of tandem repeats of D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc). The nascent polysaccharide may then be modified in a series of steps: N-deacetylation/N-sulphation of GlcNAc, epimerization of GlcA to C5 of iduronic acid (IdoA), O-sulphation at C2 of IdoA and GlcA, O-sulphation at C6 of N-sulphoglucosamine (GlcNS) and occasional O-sulphation at C3 of GlcNS. The N-deacetylation/N-sulphation, 2-O-, 6-O-and 3-O-sulphation of HS are mediated by the specific actions of HSN-deacetylase/N-sulfotransferase (HSNDST), HS 2-O-sulfotransferase (HS2ST), HS 6-O-sulfotransferase (HS6ST) and HS 3-O-sulfotransferase, respectively. In each modification step, only a fraction of the potential substrates are modified, resulting in considerable sequence diversity. This structural complexity of HS makes it difficult to determine its sequence and understand the relationship between HS structure and function.
The heparan sulfate side chain consists of alternating arrangements of D-glucuronic acid or L-iduronic acid and D-glucosamine linked by (1 → 4) glycosidic linkages. Glucosamine is typically N-acetylated or N-sulfated, and both uronic acids and glucosamine can additionally be O-sulfated. The specificity of a particular HSPG for a particular binding partner is produced by a particular pattern of carboxyl, acetyl and sulfate groups linked to glucosamine and uronic acid. In contrast to heparin, heparan sulfate contains fewer N-and O-sulfate groups and more N-acetyl groups. The heparan sulfate side chain is connected to the serine residue of the core protein via the tetrasaccharide bond linkage (-glucuronyl-beta- (1 → 3) -galactosyl-beta- (1 → 4) -xylosyl-beta-1-O- (serine)) region.
Both the heparan sulfate chains and the core protein may undergo a series of modifications that may ultimately affect their biological activity. The complexity of HS has been considered to exceed that of nucleic acids (Lindahl et al, 1998, J.biol. chem.273, 24979; Sugahara and Kitagawa, 2000, Curr. Opin. struct. biol.10, 518). The variation in HS species results from the synthesis of non-random, highly sulfated sequences of sugar residues separated by non-sulfated regions of disaccharides containing N-acetylated glucosamine. The initial conversion of N-acetylglucosamine to N-sulfoglucosamine creates a focus of other modifications, including epimerization of glucuronic acid to iduronic acid and a complex pattern of O-sulfation on glucosamine or iduronic acid. Furthermore, in the unmodified, low sulfated N-acetylated sequence, the hexuronic acid residue remains glucuronic acid, whereas in the highly sulfated N-sulfated region, the C-5 epimer iduronate predominates. This limits the number of potential disaccharide variants possible in any given chain, but does not limit the abundance of each. Most of the modification occurs in the N-sulphated domains or in regions immediately adjacent to them, such that highly sulphated regions separated by low sulphated domains are present in the mature chain (Brickman et al (1998), J.biol.chem.273(8), 4350-.
It is hypothesized that the highly variable heparan sulfate chain plays a key role in the regulation of the action of a large number of extracellular ligands (including the regulation and presentation of growth and adhesion factors to cells) through a complex combination of autocrine, and secretory and paracrine feedback loops, thereby controlling intracellular signal transduction and thus stem cell differentiation. For example, even though heparan sulfate glycosaminoglycans may be genetically described (Alberts et al (1989) Caland publishing Co., New York & London, pages 804 and 805), heparan sulfate glycosaminoglycans isolated from a single source may differ in biological activity. As shown by Brickman et al, 1998, Glycobiology "8,463, two separate pools of heparan sulfate glycosaminoglycans obtained from neuroepithelial cells can specifically activate FGF-1 or FGF-2, depending on the mitogenic status. Similarly, the ability of Heparan Sulfate (HS) to interact with FGF-1 or FGF-2 is described in WO 96/23003. According to this patent application, corresponding HS capable of interacting with FGF-1 can be obtained from murine cells at about 11 to about 13 days of embryonic day, while HS capable of interacting with FGF-2 can be obtained from about 8 to about 10 days of embryonic day.
As mentioned above, the HS architecture is highly complex and variable between HS. Indeed, changes in HS structure are thought to play an important role in promoting the distinct activities of each HS in promoting cell growth and directing cell differentiation. The structural complexity is believed to exceed that of nucleic acids, and although HS structures can be characterized as sequences of repeating disaccharide units with specific and unique sulfation patterns, no standard sequencing techniques equivalent to those of nucleic acid sequencing are currently available for determining HS sequence structures. In the absence of simple methods for determining the structure of a defined HS sequence, the skilled person confirms and structurally characterizes HS molecules by a number of analytical techniques. These include one or a combination of disaccharide analysis, tetrasaccharide analysis, HPLC, capillary electrophoresis and molecular weight determination. These analytical techniques are well known and used by those skilled in the art.
Two techniques for producing disaccharides and tetrasaccharides from HS include nitrous acid digestion and lyase digestion. The following description of one manner of performing these digestion techniques is provided by way of example only and is not intended to limit the scope of the present invention.
Nitrous acid digestion
When completed, nitrous acid based depolymerization of heparan sulfate results in the eventual degradation of the carbohydrate chain into its individual disaccharide components.
For example, nitrous acid may be prepared by the following method: separately, 250. mu.l of 0.5M H were cooled on ice2SO4And 0.5MBa (NO)2)2And 15 min. After cooling, Ba (NO)2)2And H2SO4Combined and vortexed, then centrifuged to remove the barium sulfate precipitate. 125 μ l of HNO2Adding to 20. mu.l of H2Resuspended GAG samples in O, vortexed and incubated at 25 ℃ for 15min with occasional mixing. After incubation, 1M Na2CO3Added to the sample and the sample was adjusted to
Digestion with lyase
Heparin III cleaves sugar chains on glucuronide linkages. A series of heparanases (I, II and III) each show relatively specific activity by depolymerising certain heparan sulphate sequences at specific sulphation recognition sites. Heparinase I cleaves HS chain with NS region along HS chain. This results in disruption of the sulphated domains. Heparinase III depolymerizes HS with NA domains, resulting in the separation of the carbohydrate chains into individual sulfated domains. Heparinase II cleaves primarily in the NA/NS "shoulder" domain of the HS chain, where different sulfation patterns are found. Note that: the repeating disaccharide backbone of the heparan polymer is uronic acid linked to the amino sugar glucosamine. "NS" means that the amino sugar bears a sulfate on the amino group, such that other groups at C2, C6, and C3 can be sulfated. "NA" means that the amino group is not sulfated and remains acetylated.
For example, for depolymerization in the NA region using heparinase III, in a medium containing 20mM Tris-HCL, 0.1mg/ml BSA and 4mM CaCl2The enzyme and lyophilized HS samples were prepared in the buffer of ph 7.5. Purely by way of example, heparinase III may be added at a dose of 5mU/1 μ gHS, incubated for 16h at 37 ℃ and then stopped by heating to 70 ℃ for 5 min.
Disaccharides and tetrasaccharides may be eluted by column chromatography.
Chemical synthesis of heparin or heparan sulfate oligosaccharides
Synthetic heparin or heparan sulfate oligosaccharides having a predetermined length may be prepared using conventional solution phase chemistry, and the product is purified by crystallization or flash column chromatography using silica gel 60(Fluka, gillin elm, UK). Oligosaccharides comprising 8 to 12 sugar residues can be assembled from disaccharide precursors with protecting groups. The final product can be purified by size exclusion chromatography using SephadexG-25 (Sigma-Aldrich, Gilinelm, UK) and lyophilized. The product structure and purity can be confirmed by NMR spectroscopy and mass spectrometry.
For example, the method of Cole et al (Cole CL, Hansen SU, Bar th M, Rushton G, Gardiner JM, etc. (2010) synthetic heparan sulfate oligosaccharides inhibiting endothelial cell function essential for angiogenesis, PLoS ONE 5(7) e11644.doi:10.1371/journal. pane. 0011644) can be used.
Synthetic heparin or heparan sulfate may be prepared as analogues of heparin or heparan sulfate oligosaccharides, which have been identified by size fractionation of preparations containing long-chain heparin or heparan sulfate oligosaccharides, for example commercially available preparations of heparin or heparan sulfate from e.g. porcine mucosa.
As described herein, synthetic heparin or heparan sulfate oligosaccharides can be prepared to have a specified chain length, sulfation pattern and disaccharide content, and the design of such oligosaccharides can be based on information about the chain length, sulfation pattern and disaccharide content of heparin or heparan sulfate oligosaccharides identified by analyzing the size-fractionated preparation of a heterogeneous heparin or heparan sulfate preparation.
Thus, in another aspect of the invention there is provided a method of designing heparin or heparan sulphate, optionally heparin or heparan sulphate for use in a method of treatment according to the invention, the method comprising determining one or more of the chain length, sulphation pattern, and sugar (or disaccharide) content or sequence of heparin or heparan sulphate having BMP2 binding activity.
In another aspect of the invention, there is provided a method of making, producing or preparing heparin or heparan sulphate, optionally heparin or heparan sulphate for use in the treatment methods of the invention, the method comprising one or more of the following steps:
(i) determining one or more of the chain length, sulfation pattern, and saccharide (or disaccharide) content or sequence of heparin or heparan sulfate having BMP2 binding activity;
(ii) synthesizing one or more heparin or heparan sulfate oligosaccharides having a chain length, and/or sulfation pattern and/or sugar (or disaccharide) content or sequence associated with BMP2 binding activity;
(iii) one or more heparins or heparan sulphate oligosaccharides having a chain length, and/or sulphation pattern and/or sugar (or disaccharide) content or sequence associated with BMP2 binding activity are formulated as a pharmaceutical composition or medicament.
HS3
HS3 is heparan sulfate that binds to BMP2, described in US9498494 and WO2010/030244 (referred to therein as HS/BMP2), both of which are incorporated herein by reference in their entirety.
HS3 can be obtained by a method of enriching a mixture of compounds containing one or more GAGs that bind to a polypeptide corresponding to the heparin-binding domain of
HS3 is believed to enhance (e.g., agonize) the activity of BMP-2, thereby enhancing the ability of BMP-2 to stimulate stem cell proliferation and bone formation.
HS3 may be defined functionally and structurally, in addition to being obtainable by the methods described in US9498494 and WO 2010/030244.
Functionally, HS3 is capable of binding to a polypeptide having the sequence of SEQ ID NO: 1(QAKHKQRKRLKSSCKRHP) or SEQ ID NO: 2(QAKHKQRKRLKSSCKRH) or a peptide consisting thereof, which represents the heparin-binding domain of
Preferably HS3 is also present at a K of less than 100. mu.M, more preferably less than one of 50. mu.M, 40. mu.M, 30. mu.M, 20. mu.M or 10. mu.MDBinds to BMP2 protein. Binding between HS3 and BMP2 protein can be determined by the following assay method.
BMP2 was dissolved in a blocking solution (0.2% gelatin in SAB) at a concentration of 3. mu.g/ml and a dilution series from 0-3. mu.g/ml was established in the blocking solution. 200 μ l of each BMP2 dilution was dispensed into three wells of heparin/GAG binding plates pre-coated with heparin; incubate at 37 ℃ for 2 hours, carefully wash 3 times with SAB, and add 200. mu.L of 250ng/ml biotinylated anti-BMP 2 to the blocking solution. Incubate at 37 ℃ for 1 hour, carefully wash with
In this assay, binding can be determined by measuring absorbance and can be determined relative to a control, e.g., BMP2 protein without the addition of heparan sulfate, or BMP2 protein with the addition of heparan sulfate that does not bind BMP2 protein.
In contrast to non-specific binding, binding of HS3 is preferably specific, and in this case HS3 can be identified by relating to the selection of a polypeptide having the sequence of SEQ ID NO: 1 or 2 or heparan sulfate exhibiting a high affinity binding interaction with BMP2 protein, selected from other heparan sulfates and/or GAGs.
HS3 according to the present invention preferably enhances BMP 2-induced alkaline phosphatase (ALP) activity to a greater extent in cells of the mouse myoblast cell line C2C12 than the enhancement obtained by addition of corresponding amounts of BMP2 protein alone or heparin. Preferably, it also enhances BMP 2-induced ALP activity in C2C12 cells to a greater extent than that induced by the addition of a corresponding amount of the combination of BMP2 protein and heparin, or the combination of BMP2 protein and heparan sulfate that does not bind BMP2 protein with high affinity.
The enhancement of ALP activity can be measured by performing the following ALP assay. At 37 deg.C/5% CO2Next, C2C12 cells were cultured at 20,000 cells/cm2The density of (c) was seeded in DMEM containing 10% FCS (e.g. switzerland, st. louis, MO) and antibiotics (1% penicillin and 1% streptomycin) (e.g. sigma aldrich, st. louis, MO) in 24-well plates. After 24 hours, the medium was changed to 5% FCS low serum medium containing 100ng/mL BMP2 (example)Such as R&D system, minneapolis, MN), 3mg/mL Celsus HS and different combinations of BMP2 specific (+ ve HS) and nonspecific (-ve HS) Celsus HS preparations at different concentrations. After 3 days cell lysis was performed using RIPA buffer containing 1% Triton X-100, 150mM NaCl, 10mM Tris pH 7.4, 2mM EDTA, 0.5% Igepal (NP40), 0.1% Sodium Dodecyl Sulfate (SDS) and 1% protease inhibitor cocktail set III (carlbirheim, germany). The protein content of the cell lysate was determined by using BCA protein assay kit (pierce chemical, rockford, IL). ALP activity in cell lysates was then determined by incubating the cell lysates with p-nitrophenyl phosphate substrate (invitrogen, calsbad, CA). Readings were normalized to total protein amount and expressed as relative amount to BMP2 only treated group.
Enhancement of ALP activity in C2C12 cells can also be performed by immunohistochemical techniques, for example following the following ALP staining protocol. ALP staining. C2C12 cells were cultured as described in the assay methods above. After 3 days of treatment, the cell layer was washed with PBS and stained using a leukocyte alkaline phosphatase kit (e.g., sigma-aldrich, st louis, MO) according to the manufacturer's instructions. The cell layer was fixed in citrate buffered 60% acetone and stained in an alkaline dye mixture containing naphthol AS-MX phosphatase alkaline and diazonium salt. Nuclear staining was performed using Mayer's hematoxylin solution.
These techniques can be used to identify HS3 as a heparan sulfate that enhances BMP2 protein-induced ALP activity to a greater extent in C2C12 cells than non-specific heparan sulfate (e.g., a heparan sulfate that does not bind BMP-2 protein).
HS3 prolonged the effect of BMP2 signaling at levels equal to or exceeding heparin. The evaluation can be performed by the following assay. C2C12 cells were exposed to (i) none, (ii) BMP2 only, (iii) BMP2+ heparin or (iv) BMP2+ HS372 hours, wherein the phosphorylation level of BMP 2-specific intracellular signaling molecule Smad1/5/8 was monitored by immunoblotting.
An important functional characteristic of HS3 is its ability to enhance the bone repair process, especially in mammalian subjects. This can be tested in a bone repair model in which the rate and quality of bone repair in control animals (e.g., animals not administered HS or animals that receive HS that does not bind the BMP2 protein or the peptide sequence of SEQ ID NO: 1 or 2) are compared to HS3 treated animals. The speed and quality of bone repair can be assessed by analyzing the production of bone volume over time at the wound site, e.g., analyzing the wound by X-ray and micro ct imaging.
Recent studies have shown that gamma irradiation does not affect the binding affinity of HS3 to
Structurally, N-sulfation of N-acetyl-D-glucosamine (GlcNAc) residues in HS3 has been found to be important for maintaining binding affinity to BMP2 protein. N-desulfurization has been shown to result in a significant decrease in BMP2 protein binding affinity.
6-O-sulfation of N-sulfoglucosamine (GlcNS) residues (O-sulfation at C6) was also found to be of moderate importance for maintaining binding affinity to BMP2 protein. 6-O-desulfurization results in a decrease in the binding affinity of BMP2 protein.
It was found that 2-O-sulfation of the IdoA and/or D-glucuronic acid (GlcA) residues (O-sulfation at C2) did not affect the binding of BMP2 protein. Thus, HS3 can optionally be 2-O-sulfated or 2-O-desulfurized.
The disaccharide composition of HS3 can be determined by the following steps: after digestion with heparin lyase I, II and III, the resulting disaccharide fragments were analyzed by capillary electrophoresis.
HS3 comprises heparan sulfate having a disaccharide composition within ± 10% (more preferably one of ± 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.5%) of the respective disaccharide contents shown in the table below, as determined by lyase digestion and SAX-HPLC analysis, respectively, or complete digestion with heparin lyase I, II and III, followed by capillary electrophoresis analysis of the resulting disaccharide fragments.
Disaccharides
Normalized weight percent
ΔHexUA,2SGlcNS,6S
14.8
ΔHexUA,2S-GlcNS
4.9
ΔHexUA-GlcNS,6S
11.1
ΔHexUA,2SGlcNAc,6S
4.8
ΔHexUA-GlcNS
22.2
ΔHexUA,2S-GlcNAc
1.1
ΔHexUA-GlcNAc,6S
10.1
ΔHexUA-GlcNAc
31.1
The disaccharide composition of HS3, determined by digestion to completion with heparin lyase I, II and III and then capillary electrophoresis analysis of the resulting disaccharide fragments, is likely to have a disaccharide composition of any of:
disaccharides
Normalized weight percent
ΔHexUA,2SGlcNS,6S
14.8±3.0
ΔHexUA,2S-GlcNS
4.9±2.0
ΔHexUA-GlcNS,6S
11.1±3.0
ΔHexUA,2SGlcNAc,6S
4.8±2.0
ΔHexUA-GlcNS
22.2±3.0
ΔHexUA,2S-GlcNAc
1.1±0.5
ΔHexUA-GlcNAc,6S
10.1±3.0
ΔHexUA-GlcNAc
31.1±3.0
Or
Disaccharides
Normalized weight percent
ΔHexUA,2SGlcNS,6S
14.8±2.0
ΔHexUA,2S-GlcNS
4.9±2.0
ΔHexUA-GlcNS,6S
11.1±2.0
ΔHexUA,2SGlcNAc,6S
4.8±2.0
ΔHexUA-GlcNS
22.2±2.0
ΔHexUA,2S-GlcNAc
1.1±0.5
ΔHexUA-GlcNAc,6S
10.1±2.0
ΔHexUA-GlcNAc
31.1±2.0
Or
Disaccharides
Normalized weight percent
ΔHexUA,2SGlcNS,6S
14.8±2.0
ΔHexUA,2S-GlcNS
4.9±1.0
ΔHexUA-GlcNS,6S
11.1±2.0
ΔHexUA,2SGlcNAc,6S
4.8±1.0
ΔHexUA-GlcNS
22.2±2.0
ΔHexUA,2S-GlcNAc
1.1±0.5
ΔHexUA-GlcNAc,6S
10.1±2.0
ΔHexUA-GlcNAc
31.1±3.0
Or
Disaccharides
Normalized weight percent
ΔHexUA,2SGlcNS,6S
14.8±1.0
ΔHexUA,2S-GlcNS
4.9±0.4
ΔHexUA-GlcNS,6S
11.1±1.0
ΔHexUA,2SGlcNAc,6S
4.8±0.6
ΔHexUA-GlcNS
22.2±3.0
ΔHexUA,2S-GlcNAc
1.1±0.4
ΔHexUA-GlcNAc,6S
10.1±1.0
ΔHexUA-GlcNAc
31.1±1.6
Or
Disaccharides
Normalized weight percent
ΔHexUA,2SGlcNS,6S
14.8±0.75
ΔHexUA,2S-GlcNS
4.9±0.3
ΔHexUA-GlcNS,6S
11.1±0.75
ΔHexUA,2SGlcNAc,6S
4.8±0.45
ΔHexUA-GlcNS
22.2±2.25
ΔHexUA,2S-GlcNAc
1.1±0.3
ΔHexUA-GlcNAc,6S
10.1±0.75
ΔHexUA-GlcNAc
31.1±1.2
Or
Disaccharides
Normalized weight percent
ΔHexUA,2SGlcNS,6S
14.8±0.5
ΔHexUA,2S-GlcNS
4.9±0.2
ΔHexUA-GlcNS,6S
11.1±0.5
ΔHexUA,2SGlcNAc,6S
4.8±0.3
ΔHexUA-GlcNS
22.2±1.5
ΔHexUA,2S-GlcNAc
1.1±0.2
ΔHexUA-GlcNAc,6S
10.1±0.5
ΔHexUA-GlcNAc
31.1±0.8
Digestion of HS3 with heparin lyase I, II and III and/or capillary electrophoresis analysis of disaccharides can preferably be performed as described in example 10 of US 9498494.
Digestion of HS preparations with heparin lyase may be performed as follows: HS preparations (1mg) were each dissolved in 500. mu.L of sodium acetate buffer (100mM, containing 10mM calcium acetate, pH 7.0), and 2.5mU of each of the three enzymes was added; the samples were incubated overnight (24 hours) at 37 ℃ while slightly inverting (9rpm) the sample tube; an additional 2.5mU of each of the three enzymes was added to the sample and the sample tube was gently inverted (9rpm) at 37 ℃ for an additional 48 hours; digestion was stopped by heating (100 ℃,5 min) and then freeze-dried; the digest was resuspended in 500. mu.L of water and an aliquot (50. mu.L) was taken for analysis.
Capillary Electrophoresis (CE) of the HS digested disaccharide can be performed by the following method: the capillary electrophoresis was performed by adding 20mM H3PO4To 20mM Na2HPO4·12H2O, at a pH of 3.5; column wash was 100mM NaOH (diluted from 50% w/w NaOH); both the operating buffer and the column wash were filtered using a filter unit equipped with 0.2 μm cellulose acetate membrane filter paper. Stock solutions of disaccharide Is (e.g., 12) were prepared by dissolving the disaccharide in water (1 mg/mL). The calibration curve of the standard was determined by preparing a mixture comprising all standard solutions containing 10 μ g/100 μ L of each disaccharide and preparing dilution series comprising 10,5, 2.5, 1.25, 0.625, 0.3125 μ g/100 μ L; an internal standard (. DELTA.UA, 2S-GlcNCOEt,6S) of 2.5. mu.g was included. The digest of HS was diluted with water (50. mu.L/mL) and the same internal standard (2.5. mu.g) was added to each sample. The solution was freeze dried and resuspended in water (1 mL). The sample was filtered using a PTFE hydrophilic disposable syringe filter unit.
The analysis was carried out using a capillary electrophoresis apparatus on uncoated fused silica capillaries at 25 ℃ using 20mM process buffer with a capillary voltage of 30 kV. The sample was introduced into the capillary at the cathode (reverse polarity) end using hydrodynamic injection. Prior to each run, the capillaries were rinsed with 100mM NaOH (2 min), water (2 min) and pre-treated with running buffer (5 min). The buffer replenishment system replaces the buffer in the inlet and outlet tubes to ensure that consistent volume, pH and ionic strength are maintained. Water only blanks were run at the beginning, middle and end of the sample sequence. The absorbance was monitored at 232 nm. All data is stored in the database and then retrieved and reprocessed.
Two or three replicates of digestion/analysis can be performed and the normalized percentage of disaccharides in HS digestions is calculated as the average of the analysis results.
HS3 binds to BMP2 protein or SEQ ID NO: 1 or 2 have high affinity binding.
The structural differences in HS3 compared to heparan sulfate, which does not bind BMP2 protein, can also be demonstrated by performing surface plasmon resonance analysis. For example, the angular offset can be used to distinguish HS3 from other heparan sulfates.
Fragments of HS3
Some aspects and embodiments of the invention relate to fragments of HS3, or mixtures comprising fragments of
Preferably, the fragment of HS3 is an oligosaccharide chain of HS3 that has been truncated, cleaved or separated, e.g., to produce more than one shorter chain by the action of a lyase for HS3 oligosaccharide chains. Preferred fragments are those that retain the ability to bind BMP2, enhance BMP2 mediated ALP activity, enhance BMP2 mediated Smad1/5/9 phosphorylation and/or enhance bone repair.
The mixture of HS3 fragments and HS3 fragments preferably excludes full length heparan sulfates that bind BMP2,
Full length HS3 typically has an average chain length of about 50 sugars or more, and an average molecular weight of about 15 kDa. Thus, a mixture of HS3 fragments and HS3 fragments may have less than 10% oligosaccharide chains with a chain length of greater than 50 sugars. Alternatively, the percentage may be a percentage of one selected from 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.
Mixtures of heparin or heparin sulfate oligosaccharides
The mixture may comprise a plurality of oligosaccharide chains, each of which has a defined length in terms of number or carbohydrate and optionally in terms of functional properties, such as BMP2 binding.
The mixture may be heterogeneous with respect to oligosaccharide content, e.g. it may comprise oligosaccharides as described in the present invention of variable length but all within a defined length range.
The mixture may contain other ingredients, such as other glycosaminoglycans or heparin or heparan sulfate. In some embodiments, the mixture does not comprise glycosaminoglycans, heparin or heparan sulfate other than oligosaccharides as described herein.
Formulating pharmaceutically useful compositions and medicaments
According to the present invention, there is also provided a method for the production of a pharmaceutically acceptable composition, which may be based on identified heparin or heparan sulphate. Such a production method may further comprise one or more steps selected from:
(a) identifying and/or characterizing the structure of a selected heparin or heparan sulfate;
(b) obtaining said heparin or heparan sulfate;
(c) selected heparins or heparan sulfates are mixed with pharmaceutically acceptable carriers, adjuvants or diluents.
For example, another aspect of the invention relates to a method of formulating or manufacturing a pharmaceutical composition for use in a method of treatment, the method comprising:
(i) identifying and/or isolating heparin or heparan sulfate; and/or
(ii) Pharmaceutical compositions are formulated by mixing heparin or heparan sulfate with a pharmaceutically acceptable carrier, adjuvant or diluent.
Certain pharmaceutical compositions formulated by this method may comprise a prodrug of the selected substance, wherein the prodrug is convertible in the human or animal body to the desired active agent. In other cases, the active agent may be present in the pharmaceutical composition so produced, and may be present in the form of a physiologically acceptable salt.
Biological material
The pharmaceutical compositions and medicaments of the present invention may take the form of biomaterials coated and/or impregnated with isolated heparin or heparan sulfate. The implant or prosthesis may be formed of a biomaterial. Such implants or prostheses may be surgically implanted to assist in cell transplantation.
Isolated heparin or heparan sulfate may be applied to an implant or prosthesis to accelerate new tissue formation at a desired location. It will be appreciated that heparin and heparan sulfate, unlike proteins, are particularly strong and have a better ability to withstand the solvents required for the manufacture of synthetic biological scaffolds and for application to implants and prostheses.
The biological material may be coated or impregnated with isolated heparin or heparan sulfate. The impregnation may comprise forming the biomaterial by mixing the isolated heparin or heparan sulfate with the constituent components of the biomaterial, for example in a polymerisation reaction, or absorbing the isolated heparin or heparan sulfate into the biomaterial. Coating may include adsorbing the separated heparin or heparan sulfate onto the surface of the biomaterial.
The biomaterial should allow the release of the coated or impregnated isolated heparin or heparan sulfate from the biomaterial when administered or implanted in a subject. The kinetics of biomaterial release can be altered by altering the structure, e.g., porosity, of the biomaterial.
In addition to coating or impregnating the biological material with isolated heparin or heparan sulfate, one or more bioactive molecules may be impregnated or coated on the biological material. For example, at least one selected from the group consisting of: BMP-2, BMP-4, OP-1, FGF-2, TGF-beta 1, TGF-
The biomaterial provides a scaffold or matrix support. The biomaterial may be suitable for implantation into tissue, or may be suitable for administration (e.g., as microcapsules in solution).
The implant or prosthesis should be biocompatible, e.g. non-toxic and low (most preferably non-immunogenic). The biomaterial may be biodegradable such that the biomaterial degrades. Alternatively, a non-biodegradable biomaterial may be used, with surgical removal of the biomaterial being an optional requirement.
The biomaterial may be soft and/or flexible, for example, a hydrogel, a fibrin mesh or mesh, or a collagen sponge. A "hydrogel" is a substance that forms when an organic polymer (which may be natural or synthetic) solidifies or solidifies to form a three-dimensional open lattice structure that traps water molecules or other solutions and forms a gel. Curing may occur by aggregation, coagulation, hydrophobic interactions or crosslinking.
Alternatively, the biomaterial may be a relatively rigid structure, for example formed from a solid material such as plastic or a biologically inert metal such as titanium.
The biomaterial may have a porous matrix structure that may be provided by a cross-linked polymer. The matrix is preferably permeable to nutrients and growth factors required for bone growth.
The matrix structure may be formed by cross-linking fibres such as fibrin or collagen, or by a liquid film of sodium alginate, chitosan or other polysaccharides with suitable cross-linking agents (e.g. calcium salts, polyacrylic acid, heparin). Alternatively, the scaffold may be formed as a gel, made from collagen or alginate, and crosslinked using well known methods known to those skilled in the art.
Suitable polymeric materials for matrix formation include, but are not limited to, biodegradable/bioresorbable polymers, which may be selected from: agarose, collagen, fibrin, chitosan, polycaprolactone, (DL-lactide-caprolactone) copolymer, (L-lactide-caprolactone-glycolide) copolymer, polyglycolide, polylactide, polyhydroxyalkanoate, copolymers thereof, or non-biodegradable polymers, which may be selected from: cellulose acetate, cellulose butyrate, alginate, polysulfone, polyurethane, polyacrylonitrile, sulfonated polysulfone, polyamide, polyacrylonitrile, polymethyl methacrylate, and copolymers thereof.
Collagen is a promising material for matrix construction due to its biocompatibility and advantageous properties that support cell attachment and function (U.S. Pat. No.5,019,087; Tanaka, S.; Takigawa, T.; Iichara, S. & Nakamura, T. Mechanical Properties of bioabsorbable polyglycolic acid-collagen nerve conduit "Mechanical Properties of the bioabsorbable polyglycolic acid-collagen nerve conduit" Polymer engineering & Science 2006,46,1461-. Clinically acceptable collagen sponges are one example of a matrix and are well known in the art (e.g., from intel glar life sciences).
Fibrin scaffolds (e.g., fibrin glue) provide an alternative matrix material. Fibrin glue has a wide range of clinical applications AS a wound sealant, a reservoir for the delivery of Growth factors, and AS an aid to the placement and fixation of bioimplants (Rajesh valve, Dhirendora S Katti. Growth factor delivery system for tissue engineering. materials view. Experi review in Medical devices.2006; 3. 1. 29-47; Wong C, InmanE, Spaethe R, Helgerson S.Thromb. Haemost.200389 (3):573 582; pandas, Wilson DJ. Feldman DS. fibrin scaffold AS an effective carrier for the delivery of acidic Growth factors (fibrin) in vitro Growth factor delivery system: collagen F229. collagen J. collagen delivery system for tissue engineering. collagen production in vitro tissue engineering. collagen delivery system for tissue engineering. collagen production in vivo. 12. collagen production system for tissue engineering. collagen production in vitro. collagen production of collagen production tissue engineering. 12. collagen production system for tissue engineering. collagen production of collagen production in vitro tissue engineering, collagen production system for tissue engineering, collagen production of collagen production in vivo, collagen production system, collagen production system for tissue engineering, tissue engineering and in vivo applications to collagen based materials ". biomaterials.1994; 15(9):665-672.).
Luong-Van et al (In vitro biocompatibility and bioactivity of microencapsulated heparan sulfate, "Biomaterials 28(2007)2127-2136), incorporated herein by reference, describe the prolonged local delivery of HS from polycaprolactone microcapsules.
Another example of a biomaterial is a polymer incorporating hydroxyapatite or hyaluronic acid.
Other suitable biomaterials include ceramics or metals (e.g., titanium), hydroxyapatite, tricalcium phosphate, Demineralized Bone Matrix (DBM), autografts (i.e., grafts derived from patient tissue) or allografts (grafts derived from tissue of a non-patient animal)). The biomaterial may be synthetic (e.g. metal, fibrin, ceramic) or biological (e.g. a carrier material made from animal tissue (e.g. non-human mammals (e.g. cows, pigs) or humans)).
The biological material may be supplemented with additional cells. For example, one can "seed" the biological material with stem cells (or co-synthesize it).
In one embodiment, the biomaterial may comprise a coating or impregnation with isolated heparin or heparan sulfate and further comprises BMP2 (e.g. as another component of the coating or impregnation) and cells, such as stem cells adhered to the biomaterial.
Fracture of bone
In certain aspects, the invention relates to the therapeutic use (human and veterinary) of isolated heparin or heparan sulfate in the treatment of bone fractures. Isolated heparin or heparan sulfate has been reported to enhance wound healing in bone. Isolated heparin or heparan sulfate can stimulate bone regeneration after injury and help improve wound healing of the bone. Isolated heparin or heparan sulfate may improve the rate of fracture repair, thereby reducing recovery time after injury.
Fracture is a medical condition. In the present application, "fracture" includes damage or injury to a bone, wherein the bone fractures, breaks or fractures. A fracture refers to a discontinuity in the bone. Fractures may be caused by physical shock, or mechanical stress or by medical conditions such as osteoporosis or osteoarthritis.
Orthopedic classifications of fractures include closed or open and simple or multi-fragment fractures. In closed fractures, the skin remains intact, while in open fractures, bone may be exposed through the wound site, which carries a higher risk of infection. Simple fractures occur along a single line, tending to divide the bone into two parts. Multi-fragment fractures divide a bone into multiple pieces.
Other fracture types include compression fractures, spiral fractures, full and incomplete fractures, transverse, linear and oblique fractures, and comminuted fractures.
Bone healing (fracture healing) occurs naturally in most subjects and begins after injury. Bleeding typically results in the clotting and attraction of leukocytes and fibroblasts, followed by the production of collagen fibers. This is followed by the deposition (mineralization) of the bone matrix (calcium hydroxyapatite) to convert the collagen matrix into bone. Immature regenerated bone is generally weaker than mature bone, and over time, the immature bone undergoes a remodeling process to produce mature "lamellar" bone. The complete bone healing process takes a considerable amount of time, usually several months.
Bones in which fractures occur and which may benefit from treatment with heparin or heparan sulfate oligosaccharides include all bone types, particularly all mammalian bones, including, but not limited to, long bones (e.g., femur, humerus, phalanges), short bones (e.g., carpals, tarsal bones), flat bones (e.g., skull, ribs, scapula, sternum, pelvic girdle), irregular bones (e.g., vertebrae), sesamoid bones (e.g., patella).
Bones in which fractures occur and which may benefit from treatment with heparin or heparan sulfate oligosaccharides include skeletal bone (i.e., any bone of the skeleton), bone of the cranio-facial region, bone of the axial skeleton (e.g., vertebrae, ribs), periapical bone (e.g., of the extremities), pelvic bone (e.g., pelvis).
Bones in which fractures occur and which may benefit from treatment with heparin or heparan sulfate oligosaccharides also include bones of the head (skull) and neck, including those of the face, such as the jaw, nose and cheek. In this regard, in some preferred embodiments, heparin or heparan sulfate oligosaccharides may be used to assist in the repair or regeneration of bone during dental or facial or cranial surgery, which may include the reconstruction of facial and/or oral bones (other than teeth), including, for example, the jawbone.
Bone fractures also include pathological porosity, such as that exhibited by subjects with osteoporosis.
Although not limiting to the invention, the isolated heparin or heparan sulfate oligosaccharides act primarily on cells within, adjacent to, or causing migration to the wound site, and may be on stem cells, preosteoblasts or osteoblasts, or on any accessory or angiogenic cells found within or causing migration into the wound bed.
Isolated heparin or heparan sulfate is provided, as well as pharmaceutical compositions and methods of use of medicaments comprising isolated heparin or heparan sulfate for treating bone fractures in a mammalian subject.
Treatment may include wound healing in bone. Treatment may involve repair, regeneration and growth of bone. Isolated heparin or heparan sulfate oligosaccharides promote fracture repair by promoting new bone growth. The isolated heparin or heparan sulfate oligosaccharides act to increase the rate of fracture repair, which allows the bone to heal faster, thereby shortening the recovery time of the injury. Treatment may result in improved bone strength.
Administration of heparin or heparan sulfate oligosaccharides is preferably to the tissue surrounding the fracture. This may include administration directly to fractured bone tissue. Administration may be to connective tissue surrounding a bone or fracture or to the vasculature (e.g., blood vessels) near and supplying the bone. The application may be directly to the site of injury, and may be to callus formed by initial healing of the wound.
The medicaments and pharmaceutical compositions according to the invention may be formulated for administration by a variety of routes. Most preferably, the isolated heparin or heparan sulfate is formulated in a fluid or liquid form for injection.
In some embodiments, the isolated heparin or heparan sulfate is formulated as a controlled release formulation, for example in a pharmaceutical capsule for implantation at a wound site. The isolated heparin or heparan sulfate may be attached to, impregnated into or impregnated into a support material (e.g., a biomaterial), such as a nanofiber or a biodegradable paper or textile.
Pharmaceutical compositions, medicaments, implants and prostheses comprising isolated heparin or heparan sulfate oligosaccharides may also comprise
Administration is preferably in a "therapeutically effective amount" sufficient to improve the healing of the fracture compared to the corresponding untreated fracture. The amount actually administered, the rate of administration and the time course will depend on the nature and severity of the fracture. Prescription of treatment, e.g., determination of dosage, etc., is within the responsibility of general practitioners and other medical personnel, and generally takes into account the nature of the fracture, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to practitioners. Administration of single or multiple isolated heparin or heparan sulfate doses may be performed according to the guidance of the prescribing physician. Purely by way of example, isolated heparin or heparan sulfate may be delivered at a dose of at least 1ng/ml, more preferably at least 5ng/ml, and optionally 10ng/ml or higher. The individual dose may be on the order of less than 1mg and greater than 1 μ g, for example one of about 5 μ g, about 10 μ g, about 25 μ g, about 30 μ g, about 50 μ g, about 100 μ g, about 0.5mg or about 1 mg. Examples of the above mentioned techniques and protocols can be found in Remington's Pharmaceutical sciences Lippincott, Williams & Wilkins, 20
Isolated heparin or heparan sulfate may be used to treat fractures in other treatments, such as administration of pain relief or anti-inflammatory drugs, fixation and placement of bone, such as fixation of an injured limb in a plaster model, surgical intervention, such as repositioning or moving bone to correct displacement, angulation or dislocation. If surgery is desired, isolated heparin or heparan sulfate may be administered directly to (e.g., applied to) the bone fracture during the procedure.
BMP2 protein
In this specification, BMP2 refers to bone morphogenic protein 2 (also known as bone
The amino acid sequence of bone
In the present specification, the "
The BMP2 protein may be derived, or derived, from any animal or human, e.g. a non-human animal, e.g. rabbit, guinea pig, rat, mouse or other rodent (including any from the order rodents), cat, dog, pig, sheep, goat, cow (including cow, e.g. cow, or any bovine animal), horse (including any animal of the family equines), donkey and non-human primate, or other non-human vertebrate organism; and/or a non-human mammal; and/or a human.
In the present specification, the subject to be treated may be any animal or human. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal (e.g., a rabbit, guinea pig, rat, mouse or other rodent (including cells from any rodent), cat, dog, pig, sheep, goat, bovine (including a cow, such as a cow, or any bovine animal), horse (including any equine), donkey and non-human primate). The non-human mammal may be a domestic pet, or an animal preserved for commercial purposes, such as a racehorse, or a farmed livestock such as a pig, sheep or cattle. The subject may be male or female. The subject may be a patient.
As indicated, the method according to the invention may be performed in vitro or in vivo. The term "in vitro" is intended to include procedures with cells in culture, while the term "in vivo" is intended to include procedures with intact multicellular organisms.
***
The invention includes combinations of the described aspects and preferred features unless such combinations are expressly excluded or expressly avoided.
The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.
While the invention has been described in conjunction with the exemplary embodiments outlined above, many equivalent modifications and variations will be apparent to those skilled in the art given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not restrictive. Various changes may be made to the described embodiments without departing from the spirit and scope of the invention.
For the avoidance of any doubt, any theoretical explanation provided herein is provided to enhance the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Aspects and embodiments of the present invention will now be described by way of example with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art.
All documents mentioned herein are incorporated herein by reference.
Throughout this specification, including the claims which follow, unless the context requires otherwise, the words "comprise" and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification, the singular forms "a," "an," and "the" include the plural forms in the appended claims, unless the context clearly dictates otherwise. Ranges of the invention may be expressed as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment.
All references mentioned above are incorporated herein by reference.
Brief description of the drawings
FIGS. 1A to 1I SPR competitive binding assay sensorgrams. Representative sensorgrams generated from competitive binding experiments performed by SPR. BMP-2(25nM) was pre-incubated with various heparin oligosaccharides (5. mu.g or 10. mu.g) and then coated on heparin-derived surfaces. SPR sensorgrams of heparin (A), dp4(B), dp6(C), dp8(D), dp10(E), dp12(F), de-2-O-sulfated heparin (G), de-6-O-sulfated heparin (H) and de-N-sulfated heparin (I) showed different reductions in response compared to BMP-2 alone. All sensorgrams represent three independent experiments.
FIGS. 2A to 2G chain length determine the ability of heparin to bind and stabilize BMP-2. The basic disaccharide structures (A) of heparan sulfate and chondroitin sulfate. BMP-2(20ng) was incubated with heparin-agarose with or without glycosaminoglycans (B) or size-defined heparin oligosaccharides (C) and bound BMP-2 was detected by Western blotting. (D) SPR sensorgrams show binding reactions resulting from binding of various concentrations (-6.25nM, -12.5nM, -25nM, -50nM, -100nM) of BMP-2 to heparin-coated surfaces. (E) SPR-based competitive binding assays were performed by incubating heparin oligosaccharides (10. mu.g/mL) with BMP-2(25nM) and applying it to heparin-coated SPR chips. (F, G) BMP-2 (5. mu.M) was incubated in the presence or absence of heparin oligosaccharides (50 pM; -PBS, -BMP-2 only, -heparin, -dp4, -dp6, -dp8, -dp10, -dp12) for differential scanning fluorimetry testing. The fluorescence generated by binding of SYPRO orange dye to denatured BMP-2 core was used to determine relative complex stability to BMP-2+ heparin. All data represent (B, C, D, F) or mean ± s.d. (E, G) constituting three independent experiments. P < 0.0001.
FIGS. 3A to 3H are size exclusion chromatographs of heparin oligosaccharides bound to BMP-2. (A-D) 25. mu.M of each heparin oligosaccharide (dp6, dp8, dp10 or dp12) was eluted from the Superdex200 column and detected using 232nm absorbance. (E-H) 25. mu.M BMP-2 and 25. mu.M each heparin oligosaccharide (dp6, dp8, dp10 or dp12) were eluted from the Superdex200 column and detected using an absorbance at 232 nm. The chromatographic analysis represents three independent experiments.
FIGS. 4A to 4E BMP-2 induced phosphorylation of Smad1/5/9, osteogenic gene transcription and ALP activity of heparin oligosaccharides. (A) C2C12 cells were stimulated for up to 72 hours with or without BMP-2(100ng/mL) and heparin oligosaccharides (dp4, dp6, dp8, dp10 and dp 12; 5. mu.g/mL) and then assayed for Smad1/5/9 phosphorylation by Western blotting. C2C12 cells were stimulated with BMP-2 for 3 days in the presence or absence of heparin or heparin oligosaccharides, and then osteogenic gene transcription (B-D) or ALP activity (E) was determined. Data are presented as mean ± standard error of three independent experiments. P <0.0001, p <0.001, p <0.01, ns-p > 0.05.
FIGS. 5A to 5E BMP-2 binding to desulfated heparin and thermal stability. (A) Structure of the main repeating disaccharide unit in heparin/HS chain. (B) BMP-2(20ng) competes with heparin sepharose bead binding. (C) SPR-based competitive binding assays were performed by incubating selectively desulfated heparin (10. mu.g/mL) with BMP-2(25nM) and applying it to heparin-coated SPR chips. (D-E) differential scanning fluorometric analysis was performed by incubating BMP-2 (5. mu.M) with or without selective desulfated heparin (50. mu.M) (-PBS, -BMP-2 only, -heparin, -de-2-O-sulfation, -de-6-O-sulfation, -de-N-sulfation) and SYPRO orange dye at elevated temperature. The fluorescence generated by binding of SYPRO orange dye to denatured BMP-2 core was used to determine relative complex stability to BMP-2+ heparin. Data represent (B, D) or mean ± s.d. (C, E) constituting three independent experiments. P < 0.0001; ns-p > 0.05.
FIGS. 6A to 6E BMP-2 induced phosphorylation of Smad1/5/9, transcription of osteogenic genes and ALP activity of selectively desulfated heparins. (A) C2C12 cells were stimulated for up to 72 hours with or without BMP-2(100ng/mL) and heparin or specific desulfated heparin (5. mu.g/mL). Smad1/5/9 phosphorylation was then detected by Western blotting. (B-E) C2C12 cells were stimulated and cultured for 3 days in the presence or absence of heparin or desulphated heparin (5. mu.g/mL) with or without BMP-2(100ng/mL), and then osteogenic gene transcription (B-D) or ALP activity (E) was measured. Data are presented as mean ± standard deviation of three independent experiments. P <0.0001, p <0.001, ns-p > 0.05.
Figures 7A to 7B BMP-2 induced osteoblast differentiation and mineralization with heparin oligosaccharide and optionally desulphated heparin. C2C12 cells were stimulated with or without BMP-2(100ng/mL) and heparin, heparin oligosaccharides or specific desulfated heparin (5. mu.g/mL) for 12 days. (A) Cells were stained with alizarin red to detect the presence of calcium. (B) Alizarin red was extracted and quantified using spectrophotometry (absorbance at 405 nm) and normalized to individual BMP-2 treated groups. Data represent (a) or represent (B) the mean ± s.d.. p <0.0001, ns-p >0.05 of three independent experiments.
Figures 8A to 8C establish controls for rat ectopic bone formation assays. (A) Representative digital images of samples taken from rat hind limb muscles, 2D X radiation and 3D μ -CT micrographs. Collagen sponge treatment alone (control; n-4); or 5 μ g of BMP-2 (BMP; n ═ 4); or 5 μ g BMP-2+25 μ g heparin (Hep; n ═ 4) was used. (B) Bone volume measurements (mm3) for each sample were determined by μ -CT analysis. Results are expressed as mean ± s.e.m. (C) Representative histological sections show the absence/presence of calcified bone matrix in the harvested samples. Staining included hematoxylin/eosin, modified four-color (blue: osteoid, red: bone) and von Kossa/McNeal staining (black: calcified deposits). BM: bone marrow, B: bone, C: calcified matrix with
Figures 9A to 9C dps8, 10 and 12 were evaluated in a rat ectopic bone formation assay. (A) Representative digital images of samples taken from rat hind limb muscles, 2D X radiation and 3D μ -CT micrographs. Treatment was with collagen sponges containing 5 μ g BMP-2 and 25 μ g dps8 (n-4), 10 (n-4) or 12 (n-5). (B) Bone volume measurements (mm3) for each sample were determined by μ -CT analysis (dashed line represents bone volume for heparin treated group). Results are expressed as mean ± s.e.m. (C) Representative histological sections show the presence of calcified bone matrix in the sample. Staining included hematoxylin/eosin, modified four-color (blue: osteoid, red: bone) and von Kossa/McNeal staining (black: calcified deposits). BM: bone marrow, B: bone, C: calcified matrix with
FIGS. 10A to 10C des-2-O-, des-6-O-and des-N-sulfated heparins were evaluated in a rat ectopic bone formation assay. (A) Representative digital images of samples taken from rat hind limb muscles, 2D X radiation and 3D μ -CT micrographs. Treatment was with collagen sponges containing 5 μ g BMP-2 and 25 μ g des-2-O- (N ═ 4), des-6-O- (N ═ 4) or des-N-sulfated (N ═ 5) heparin. (B) Bone volume measurements (mm3) for each sample were determined by μ -CT analysis (dashed line represents bone volume for heparin treated group). Results are expressed as mean ± s.e.m. (C) Representative histological sections showed the presence of calcified bone matrix in the harvested samples. Staining included hematoxylin/eosin, modified four colors (blue: osteoid, red: bone) and von Kossa/McNeal staining (black: calcified deposits). BM: bone marrow, B: bone, C: calcified matrix with
FIGS. 11A to 11E binding, stabilization and activation of BMP-2 by other sulfated polysaccharides. (A) Representative SPR sensorgrams show the binding reactions generated by various sulfated polysaccharides (10. mu.g/mL) preincubated with BMP-2(25nM) and applied to heparin-derived surfaces. (B) Normalized SPR data from (a) indicating the percentage of BMP-2 sequestered into solution by the various sulfated polysaccharides. (C) Differential scanning fluorescence was performed by incubating BMP-2 (5. mu.M) in the presence or absence of various sulfated polysaccharides (50. mu.M). (D) The fluorescence generated by binding of SYPRO orange dye to denatured BMP-2 core was used to determine relative complex stability to BMP-2+ heparin. (E) C2C12 cells were treated with BMP-2(100ng/mL) with or without various sulfated polysaccharides (5. mu.g/mL) for 3 days, and then proteins were extracted and ALP activity was evaluated. All data represent (a, C) or mean ± s.d. (B, D-E) constituting three independent experiments. P <0.01, p <0.001, p < 0.0001.
FIGS. 12A to 12E binding, stabilization and activation of BMP-2 by de-N-sulfated/re-N-acetylated dp 12. (A) Representative SPR sensorgrams show binding reactions generated by pre-incubation of heparin, heparin dp12 or de-N-sulfated/re-N-acetylated heparin dp12 (10. mu.g/mL) with BMP-2(25nM) and application to heparin-derived surfaces. (B) The normalized SPR data from (a) represents the percentage of BMP-2 sequestered into solution by heparin, heparin dp12 or desulphated/re-N-acetylated heparin dp 12. (C) Differential scanning fluorescence method was performed by incubating BMP-2(5 μ M) in the presence or absence of heparin, heparin dp12 and de-N-sulfated/heavy-N-acetylated heparin dp12(50 mM). (D) The fluorescence generated by binding of SYPRO orange dye to denatured BMP-2 core was used to determine relative complex stability to BMP-2+ heparin. (E) C2C12 cells were treated with BMP-2(100ng/mL) in the presence or absence of heparin, heparin dp12 or desulphated/re-N-acetylated heparin dp12(5 μ g/mL) for 3 days, after which proteins were extracted and ALP activity was assessed. All data represent (a, C) or mean ± s.d. (B, D-E) constituting three independent experiments. P <0.001, p <0.0001, ns-p > 0.05.
Fig. 13 the peaks relate to large and small fractions separated and combined independently.
Fig. 14 relative ALP activity of heparin fragments and HS3 fragments of different lengths.
Figure 15 sulphation analysis of HS3 fragment. The relative compositions of NAc disaccharide, N-sulfated disaccharide, 6-O-sulfated disaccharide and 2-O-sulfated disaccharide in HS3 fragments of different sizes are shown.
Figure 16 shows in tabular form the disaccharide composition differences between HS3 fragments of different lengths.
Figure 17 shows the disaccharide composition of HS3 fragments with different lengths.
FIG. 18 heparin dp12 and HS3>dp36Sulfation comparison between. Heparin dp12 and HS3>dp36Is the most biologically active fragment derived from heparin and
FIG. 19 heparin dp12 and HS3>dp36Comparison of disaccharide composition (the most biologically active fragment derived from heparin and HS 3).
Figure 20 disaccharide composition comparison between heparin dp12 and HS3> dp 36. These are the most biologically active fragments of heparin and HS3, and have different disaccharide compositions, as shown in the table.
Examples
In the following examples, the inventors describe the production of heparan sulfate and heparin oligosaccharides with variable size. In addition, the inventors demonstrate that heparan sulfate and heparin oligosaccharide fragments with reduced chain length can function as effectively as full-length molecules.
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