N-terminal derivatisation of proteins with polysaccharides

• US 9,492,556 B2
• Filed: 02/04/2015
• Issued: 11/15/2016
• 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, Laing, Peter, Gregoriadis, Gregory
• Primary Examiner(s): Cordero Garcia, Marcela M
• Application Number:
  US14/614,406
• Publication Number:
  US 20150147307A1
• Time in Patent Office: 650 Days
• Field of Search: None
• US Class Current: 1/1
• 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 methods for producing N-terminal derivatives of proteins in which a polysaccharide, preferably having at least terminal sialic units, and preferably consisting essentially only of sialic acid units, is reacted at the N-terminus of a protein or peptide under controlled conditions to produce an N-terminal derivative. The controlled conditions include use of acidic pH for the derivatization step and a higher pH for purification. The derivatives are useful for improving pharmacokinetics and pharmacodynamics of proteins and peptides.

Claims

1. A composition comprising a population of polysialic acid derivatives of a DNase protein, wherein the derivatives comprise between 2 and 200 sialic acid units and wherein at least 85% of the population is derivatised with polysialic acid at the N-Terminal amine only.

2. The composition of claim 1, wherein at least 90% of the population is derivatised with polysialic acid at the N-Terminal amine only.

3. The composition of claim 1, wherein the DNase is DNase I.

4. The composition of claim 1, wherein the derivatives comprise 2-120 sialic acid units.

5. The composition of claim 1, further comprising one or more pharmaceutically acceptable excipients.

6. The composition of claim 1, wherein the polysialic acid is linked to the protein via a linker.

7. The composition of claim 1, wherein the derivatives has a polydispersity of less than 1.3.

19. A method for treating cystic fibrosis, lupus, or a tumor comprising administering the composition of claim 1 to a patient in need thereof.

8. A polysialic derivative according to formula (I)

9. The composition of claim 8, wherein the DNase is DNase I.

10. The composition according to claim 8, wherein the derivatives comprise 2-120 sialic acid units.

11. The composition according to claim 8, further comprising one or more pharmaceutically acceptable excipients.

12. A method for producing the purified N-terminal derivative of a DNase protein of claim 8, comprising reacting (i) a Polysialic acid (PSA) with DNase at the amine group of the N-terminus of the DNase in aqueous acidic solution to produce an N-terminal derivative;

and (ii) purifying the resultant N-terminal derivative in an aqueous solution of higher pH than in step (i) to obtain a population of derivatives comprising between 2 and 200 sialic acid units and wherein at least 85% of the population is derivatised with polysialic acid at the N-Terminal amine only.

13. The method of claim 12, wherein the DNase is DNase I.

14. The method according to claim 12, wherein the PSA comprises at least one sialic acid unit or moiety derived from a sialic acid unit.

15. The method according to claim 12, wherein the PSA comprises a sialic acid unit or moiety derived from a sialic acid unit at its non-reducing and/or reducing end.

16. The method according to claim 12, wherein the PSA consists substantially only of units of sialic acid.

17. The method according to claim 12, wherein the pH of the aqueous solution in step (i) is in the range 4.0-6.0 and in step (ii) is in the range 6.5-8.5.

18. An N-terminal derivative of DNase which is obtainable by a method according to claim 12.