NOVEL HEPARIN ENTITIES AND METHODS OF USE

• US 20110065085A1
• Filed: 10/14/2009
• Published: 03/17/2011
• Est. Priority Date: 09/17/2009
• Status: Abandoned Application

• Current Assignee: W. L. Gore & Associates (W.L. Gore & Associates)
• Original Assignee: W. L. Gore & Associates (W.L. Gore & Associates)
• Inventors: Drumheller, Paul D., Biran, Roy, Claude, Charles D., Cleek, Robert L.
• Application Number:
  US12/579,308
• Publication Number:
  US 20110065085A1
• Time in Patent Office: Days
• US Class Current: 435/4
• CPC Class Codes: A61L 2300/236 Glycosaminoglycans, e.g. heparin, hyaluronic acid, chondroitin
• CPC Class Codes: A61L 2300/42 Anti-thrombotic agents, anticoagulants, anti-platelet agents
• CPC Class Codes: A61L 27/54 Biologically active materials, e.g. therapeutic substances A61L27/227 takes precedence
• CPC Class Codes: A61L 29/16 Biologically active materials, e.g. therapeutic substances A61L29/048 takes precedence
• CPC Class Codes: A61L 31/022 Metals or alloys
• CPC Class Codes: A61L 31/048 obtained by reactions only involving carbon-to-carbon unsaturated bonds A61L31/041 takes precedence
• CPC Class Codes: A61L 31/10 Macromolecular materials
• CPC Class Codes: A61L 31/16 Biologically active materials, e.g. therapeutic substances A61L31/047 takes precedence
• CPC Class Codes: A61L 33/0011 Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
• CPC Class Codes: A61L 33/08 Polysaccharides
• CPC Class Codes: A61L 33/128 Other specific proteins or polypeptides not covered by A61L33/122 - A61L33/126
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C08L 27/12 containing fluorine atoms

Abstract

The present invention relates to immobilized biologically active entities that retain a significant biological activity following manipulation. The invention also comprises a medical substrate comprising a heparin entity bound onto a substrate via at least one heparin molecule, wherein said bound heparin entity is heparinase-1 sensitive.

Claims

1. A method of determining the structure of a heparin entity bonded to a substrate, comprising the steps of:

a) providing a substrate comprising a heparin entity,

b) depolymerizing the heparin entity to generate a mixture of soluble heparin fragments,

c) detecting each heparin fragment in said mixture using column chromatography,

d) determining the identity of each detected heparin fragment from step (c), and

e) deriving the structure of the heparin entity from the identities of the detected heparin fragments.

2. The method of claim 1, wherein said depolymerizing is by heparinase-1 depolymerization.

3. The method of claim 1, wherein said column chromatography is SAX-HPLC.

4. A method of determining the structure of a heparin entity bonded to a substrate, comprising the steps of:

a) providing a substrate comprising a heparin entity,

b) depolymerizing the heparin entity to generate a surface comprising surface-bonded heparin fragments,

c) reacting the surface with a labeling reagent which introduces a detectable component to said surface-bonded heparin fragments,

d) detecting said surface-bonded heparin fragments via said detectable component,

and

e) deriving the structure of the heparin entity from the presence of the surface-bonded heparin fragments.

5. The method of claim 4, wherein said depolymerizing is by heparinase-1 depolymerization.

6. The method of claim 4, wherein said labeling reagent is a lanthanoid Michaels-like addition organo-complex.

7. The method of claim 4, wherein said organo-complex is terbium tris(4-methylthio)benzoate.

8. The method of claim 4, wherein said labeling reagent is a metachromatic dye.

9. The method of claim 4, wherein said metachromatic dye is toluidine blue.

10. The method of claim 4, wherein said detecting is by epifluoroscence, absorption spectroscopy and visually.

11. A method of determining the spatial distribution of a heparin entity bonded to a substrate, comprising the steps of:

a) providing a substrate comprising a heparin entity,

b) depolymerizing the heparin entity to generate a surface comprising surface-bonded heparin fragments,

c) reacting the surface with a labeling reagent which introduces a detectable component to said surface-bonded heparin fragments,

d) detecting said surface-bonded heparin fragment via said detectable component,

and

e) deriving the spatial distribution of the heparin entity from the presence of the surface-bonded heparin fragments.

12. The method of claim 11, wherein said depolymerizing is by heparinase-1 depolymerization.

13. The method of claim 11, wherein said labeling reagent is a lanthanoid Michaels-like addition organo-complex.

14. The method of claim 11, wherein said labeling reagent is terbium tris(4-methylthio)benzoate.

15. The method of claim 11, wherein said organo-complex comprises chemisorbed gold nanoparticles.

16. The method of claim 11, wherein said detecting is by epifluoroscent microscopy or transmission electron microscopy.

17. A system for determining the structure of a heparin entity bonded to a substrate, comprising:

a) a depolymerization solution,

b) a labeling reagent solution, and

c) a detector.

18. The system of claim 17, wherein said depolymerization solution comprises heparinase-1.

19. The system of claim 17, wherein said labeling reagent solution comprises toluidine blue, and terbium tris(4-methylthio)benzoate.

20. The system of claim 17, wherein said detector comprises SAX-HPLC, an epifluoroscent microscope, and an absorption spectroscope.