N-terminal polysialylation

• US 10,300,144 B2
• Filed: 07/25/2007
• Issued: 05/28/2019
• 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: Jain, Sanjay, Zhang, Rongsheng
• Primary Examiner(s): Audet, Maury A
• Application Number:
  US12/375,010
• Publication Number:
  US 20100022441A1
• Time in Patent Office: 4,325 Days
• Field of Search: None
• 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 method for producing a population of amino-terminal polysialic acid derivatives of insulin the method comprising:

performing a derivatization reaction in a first aqueous solution at pH 6.0 which couples a polysialic acid comprising 2-125 sialic acid units to only the amino-terminal amine of the B-chain of the insulin, thereby producing the population of amino-terminal polysialic acid derivatives of insulin; and

purifying the population of amino-terminal polysialic acid derivatives of insulin using a chromatographical method and eluting in a second aqueous solution at pH 7.4,

wherein at least 85% of the population of amino-terminal polysialic acid derivatives of insulin consists of a polysialic acid coupled only at the amino-terminal amine of the B-chain of the insulin.

2. The method according to claim 1, wherein the polysialic acid has a reactive aldehyde group which reacts with the insulin and the coupling reaction is carried out under reducing conditions.

3. The method according to claim 2, wherein the reactive aldehyde group is at the non-reducing end of the polysialic acid.

4. The method according to claim 2, wherein the reactive aldehyde is at the reducing end of the polysialic acid and the non-reducing end has been passivated such that it does not react with the insulin.

5. The method according to claim 1, wherein the polysialic acid 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.

6. The method according to claim 1, which is carried out in the presence of a formulation additive.

7. The method according to claim 6, wherein the formulation additive is selected from the group consisting of one or more buffers, stabilizers, surfactants, salts, polymers, metal ions, sugars, polyols and amino acids.

8. The method according to claim 1, wherein the derivatization reaction includes a bifunctional reagent with at least one functional group comprising N-maleimide, vinylsulphone, N-iodoacetamide, orthopyridyl group or N-hydroxysuccinimide.

9. The method according to claim 1, wherein the derivatization reaction is performed under reducing conditions.

10. The method according to claim 9, wherein the derivatization reaction includes hydrogen with catalysts, a hydride, L-ascorbic acid, sodium metabisulphite or L-selectride.

11. The method according to claim 10, wherein the hydride is an alkali metal hydride.

12. The method according to claim 1, wherein the polysialic acid is α

-2,8 linked polysialic acid.

13. The method according to claim 1, wherein the polysialic acid comprises 10-80 sialic acid units.

14. The method according to claim 13, wherein the polysialic acid comprises 20-60 sialic acid units.

15. The method according to claim 1, wherein the polysialic acid has a weight average molecular weight in the range about 1 kDa to about 35 kDa.

16. The method according to claim 15, wherein the polysialic acid has a weight average molecular weight in the range about 10 kDa to about 20 kDa.

17. The method according to claim 1, wherein the insulin is derivatised by the polysialic acid at the reducing terminal unit of the polysialic acid.

18. The method according to claim 1, wherein the population of amino-terminal polysialic acid derivatives of insulin has a polydispersity that is less than 1.3.

19. The method according to claim 1, wherein the chromatographical method comprises hydrophobic interaction chromotography, size exclusion chromotography, high performance liquid chromatography or ion exchange chromatography.

20. The method according to claim 1, wherein each amino-terminal polysialic acid derivative of insulin has the general formula (I):

21. The method according to claim 20, wherein L is a bond or is:

22. The method according to claim 20, wherein m is an integer from 10 to 80.

23. The method according to claim 1, wherein the population of amino-terminal polysialic acid derivatives of insulin is mixed with one or more pharmaceutically acceptable excipients to produce a pharmaceutical composition.