N-TERMINAL POLYSIALYLATION

• US 20100022441A1
• Filed: 07/25/2007
• Published: 01/28/2010
• Est. Priority Date: 07/25/2006
• Status: Active Grant

• Current Assignee: Lipoxen Technologies Limited (Xenetic Biosciences, Inc.)
• Original Assignee: Lipoxen Technologies Limited (Xenetic Biosciences, Inc.)
• Inventors: Zhang, Rongsheng, Jain, Sanjay
• Granted Patent: US 10,300,144 B2
• Time in Patent Office: Days
• US Class Current: 514/1.100
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/1816 Erythropoietin [EPO]
• CPC Class Codes: A61K 38/193 Colony stimulating factors [CSF]
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: A61K 47/61 the organic macromolecular compound being a polysaccharide or a derivative thereof
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/06 of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
• CPC Class Codes: A61P 5/50 for increasing or potentiating the activity of insulin
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: C07K 14/575 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/21001 : Deoxyribonuclease I (3.1.21.1)
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention relates to a composition comprising a population of polysaccharide derivatives of a protein, wherein the protein is insulin or an insulin-like protein and the polysaccharide is anionic and comprises between 2 and 125 saccharide units, and wherein the population consists of substantially only N-terminal derivatives of the protein. Typically, the polysaccharide is PSA. The present invention also relates to pharmaceutical compositions comprising the novel compounds, and methods for making the novel compounds.

Claims

1. A composition comprising a population of polysaccharide derivatives of a protein, wherein the protein is insulin or an insulin-like protein and the polysaccharide is anionic and comprises between 2 and 125 saccharide units, and wherein the population consists substantially only of N-terminal derivatives of the protein.

2. A composition according to claim 1 wherein the polysaccharide is selected from the group consisting of polysialic acid, heparin, hyaluronic acid and chondroitin sulphate.

3. A composition according to claim 2 wherein the polysaccharide is polysialic acid.

4. A composition according to claim 1 wherein the insulin or insulin-like protein is derivatised by the polysaccharide at the reducing terminal unit of the polysaccharide.

5. A composition according to claim 1 wherein the polysaccharide derivatives are of formula (I)

wherein m is at least one;

HNB is derived from B—

NH₂ which is the N-terminus insulin or an insulin-like protein;

L is a bond, a linking group, or comprises a polypeptide or a synthetic digomer;

GlyO is an anionic saccharide unit;

wherein the linking group, if present, is of the formula

—

Y—

C(O)—

R¹—

C(O)—

;

wherein R¹ is a difunctional organic radical selected from the group consisting of alkanediyl, arylene, alkarylene, heteroarylene and alkylheteroarylene, any of which may substituted and/or interrupted by carbonyl, ester, sulfide, ether, amide and/or amine linkages; and

Y is NR² or NR²—

NR² wherein R² is H or C_1-6 alkyl.

6. A composition according to claim 5 wherein L is a bond or is

7. A composition according to claim 1 wherein the polysaccharides comprise 10-80 sialic acid units.

8. A composition according to claim 1 wherein the polydispersity of the anionic polysaccharide is less than 1.3.

9. A composition according to claim 1 which is a pharmaceutical composition and comprises one or more pharmaceutically acceptable excipients.

10. (canceled)

11. A method for producing a polysaccharide derivative of insulin or of an insulin-like protein which method comprises coupling an anionic polysaccharide comprising 2-125 saccharide units to only the N-terminal amine of the insulin or insulin-like protein.

12. A method according to claim 11 wherein the anionic polysaccharide has a reactive aldehyde group which reacts with the insulin or insulin-like protein and the derivatisation reaction is carried out under reducing conditions.

13. A method according to claim 12 wherein the reactive aldehyde group is at the non-reducing end of the polysaccharide.

14. A method according to claim 12 wherein the reactive aldehyde is at the reducing end of the polysaccharide and the non-reducing end has been passivated such that it does not react with the insulin or insulin-like protein.

15. A method according to claim 11 wherein the polysaccharide reacts with an amine group of the insulin or insulin-like protein.

16. A method according to claim 15 where the amine is a terminal amine group.

17. A method according to claim 15 wherein the amine is derived from a lysine amino acid of the insulin or insulin-like protein.

18. A method according to claim 11 wherein the anionic polysaccharide has a reactive aldehyde group and the method comprises

(a) converting said aldehyde group to an amine,

(b) reacting said amine with a bifunctional reagent comprising at least one functional group selected from N-maleimide, vinylsulphone, N-iodoacetamide, orthopyridyl group and N-hydroxysuccinimide, to form a reaction intermediate;

and

(c) reacting the reaction intermediate with the insulin or insulin-like protein.

19. A method according to claim 11 wherein the anionic polysaccharide reacts with a terminal amine group of the insulin or insulin-like protein in a first aqueous solution of acidic pH;

and the resultant polysaccharide derivative is purified in a second aqueous solution of higher pH than the first aqueous solution.

20. A method according to claim 19 wherein the pH of the first aqueous solution is in the range 4.0-6.0 and the pH of the second aqueous solution is in the range 6.5-8.5.

21. A method according to claim 11 which is carried out in the presence of a formulation additive.

22. A method according to claim 21 wherein the formulation additive is selected from the group consisting of one or more buffers, stabilisers, surfactants, salts, polymers, metal ions, sugars, polyols and amino acids.

23. A method according to claim 22 wherein the formulation additive is sorbitol, trehalose or sucrose.

24. A method according to claim 22 wherein the formulation additive is a non-ionic surfactant.

25. A method according to claim 22 wherein the formulation additive is a polymer which is PSA, PEG or hydroxy-beta-cyclodextrin.

26. A method according to claim 22 wherein the formulation additive is a divalent metal ion.

27. A method according to claim 22 wherein the formulation additive is a sodium phosphate buffer.

28. A polysaccharide derivative of insulin or of an insulin-like protein obtainable by a method according to claim 11.

29. A method according to claim 18 wherein the anionic reaction intermediate reacts with a terminal amine group of the insulin or insulin-like protein in a first aqueous solution of acidic pH;

and the resultant polysaccharide derivative is purified in a second aqueous solution of higher pH than the first aqueous solution.

30. A method according to claim 29 wherein the pH of the first aqueous solution is in the range 4.0-6.0 and the pH of the second aqueous solution is in the range 6.5-8.5.