Non-anticoagulant polysaccharide compositions
Preparations of polysaccharides lacking substantial anticoagulant activity are provided herein. Methods of making and using such preparations are provided.
1. A pharmaceutical composition comprising a low molecular weight heparin preparation and a pharmaceutically acceptable carrier, wherein the low molecular weight heparin preparation is produced by a process comprising digesting unfractionated heparin (UFH) with a heparinase that cleaves at the pentasaccharide site comprising:
(1) digesting UFH with Bacteroides thetaiotaomicron Heparinase I;
(2) monitoring absorbance at 232 nm during the step of digesting; and
(3) quenching the digestion reaction when the absorbance at 232 nm reaches between 1.7 and 2.0, wherein the low molecular weight heparin preparation has an anti-Xa activity of 10-40 IU/mg and an anti-IIa activity of 5-15 IU/mg, a weight average molecular weight between 3,500 and 5,500 and a molecular weight distribution such that 30-50% of the oligosaccharides of the preparation have a molecular weight <3000 Da; 40-55% of the oligosaccharides have a molecular weight between 3000-8000 Da, and 5-30% of the oligosaccharides have a molecular weight >8000 Da, wherein the preparation has no glycol split uronic acid residues, and wherein the preparation consists essentially of polysaccharides of Formula I:
U 2S —H x,y,z —[U w —H x,y,z ] n
wherein n is an integer such that n=1-20;
w=−2OS or −2OH;
x=—NS or —NAc;
y=−3OS or −3OH;
z=−6OS or −6OH;
wherein U indicates a uronic acid residue and H indicates a hexosamine residue;
and wherein w, x, y, and z are independently the same or different on each U or H residue.
2. The pharmaceutical composition of claim 1 , wherein the Heparinase I is present at about 1.0 to 5.0 IU/g UFH.
3. The pharmaceutical composition of claim 1 , wherein the step of digesting does not include treatment with a thiol reducing agent.
4. The pharmaceutical composition of claim 1 , wherein the preparation has an anti-Xa activity less than 40, 30, or 20 IU/mg.
5. The pharmaceutical composition of claim 1 , wherein the low molecular weight heparin preparation has an anti-IIa activity of 5 to less than 10 IU/mg.
6. The pharmaceutical composition of claim 1 , wherein the preparation has polysaccharide chains having greater than 40% U 2S H NS,6S disaccharide residues.
7. The pharmaceutical composition of claim 1 , wherein the preparation has polysaccharide chains having greater than 50% U 2S H NS,6S disaccharide residues and a degree of desulfation less than 40%.
8. The pharmaceutical composition of claim 7 , wherein the polysaccharides of the preparation have greater than 50% U 2S H NS,6S disaccharide residues.
9. The pharmaceutical composition of claim 7 , wherein the polysaccharides of the preparation have greater than 60% U 2S H NS,6S disaccharide residues.
10. The pharmaceutical composition of claim 7 , wherein the polysaccharides of the preparation have greater than 70% U 2S H NS,6S disaccharide residues.
11. The pharmaceutical composition of claim 7 , wherein the polysaccharides of the preparation have greater than 80% U 2S H NS,6S disaccharide residues.
12. The pharmaceutical composition of claim 7 , wherein the polysaccharides of the preparation have a degree of desulfation less than 30%.
13. The pharmaceutical composition of claim 12 , wherein the polysaccharides of the preparation have a degree of desulfation less than 20%.
14. The pharmaceutical composition of claim 13 , wherein the polysaccharides of the preparation have a degree of desulfation less than 10%.
15. The pharmaceutical composition of claim 1 , wherein less than 2% of the polysaccharide chains of the preparation comprise the tetrasaccharide UH NAc,6S GH NS,3S,6S .
16. The pharmaceutical composition of claim 1 , wherein about 20-30% of the polysaccharide chains of the preparation comprise the pentasaccharide H NAc,6S GH NS,3S,6S I 2S H NS,6S .
17. The pharmaceutical composition of claim 1 , wherein about 20-30% of the polysaccharide chains of the preparation comprise the tetrasaccharide U 2S H NAc,6S GH NS,3S,6S .
18. The pharmaceutical composition of claim 1 , wherein the preparation has a polydispersity of about 1.6 to 2.1.
19. The pharmaceutical composition of claim 1 , wherein the preparation has anti-metastatic activity.
20. The pharmaceutical composition of claim 1 , wherein the preparation binds to one or more of: VEGF, FGF, SDF-1, or P-selectin.
21. The pharmaceutical composition of claim 20 , wherein the preparation inhibits an activity of one or more of: VEGF, FGF, SDF-1, or P-selectin.