# US 9,040,478 B2 · Derivatisation of granulocyte colony-stimulating factor > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,040,478 B2 - Document ID: US9040478B2 - Status: Granted - Kind: B2 - Application number: 13/646,584 - Filed: 2012-10-05 - Published: 2014-06-12 - Granted: 2015-05-26 - Earliest claimed: 2006-07-25 - Assignees: Lipoxen Technologies Limited - Applicants: Lipoxen Technologies Limited - Inventors: Sanjay Jain; Peter Laing; Gregory Gregoriadis - Web page: https://patentgrep.com/patent/US9040478B2 ## CPC - A61K 47/61: the organic macromolecular compound being a polysaccharide or a derivative thereof - A61K 38/1816: {Erythropoietin [EPO]} - A61K 38/193: {Colony stimulating factors [CSF]} - A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases} - A61P 3/04: Anorexiants; Antiobesity agents - A61P 3/10: for hyperglycaemia, e.g. antidiabetics - A61P 5/06: of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH - A61P 5/50: for increasing or potentiating the activity of insulin - A61P 7/00: Drugs for disorders of the blood or the extracellular fluid - A61P 7/06: Antianaemics - A61P 11/00: Drugs for disorders of the respiratory system - A61P 13/12: of the kidneys - A61P 35/00: Antineoplastic agents - A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - C07K 14/505: Erythropoietin [EPO] - C07K 14/52: Cytokines; Lymphokines; Interferons - C07K 14/535: Granulocyte CSF; Granulocyte-macrophage CSF - C07K 14/575: Hormones (derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695) - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - C12Y 301/21001: Deoxyribonuclease I (3.1.21.1) - 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) - 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 - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES - Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE ## Abstract The present invention relates to a compound which is a polysaccharide derivative of GCSF, or of a GCSF like protein, wherein the polysaccharide is anionic and comprises between 2 and 200 saccharide units. The present invention also relates to pharmaceutical compositions comprising the novel compounds, and methods for making the novel compounds. ## Claims ### Claim 1 A compound which is an amino-terminal polysaccharide derivative of granulocyte colony-stimulating factor (GCSF), wherein the polysaccharide is attached to the amino terminus of the GCSF, wherein the polysaccharide is anionic and comprises between 2 and 200 saccharide units. ### Claim 2 (depends on 1) The compound according to claim 1 wherein the polysaccharide is selected from polysialic acid, heparin, hyaluronic acid or chondroitin sulphate. ### Claim 3 (depends on 1) The compound according to claim 1 wherein the GCSF is derivatised by the polysaccharide at the reducing terminal unit of the polysaccharide. ### Claim 4 A compound of formula (I) wherein m is at least one; XB is B—XH wherein B is GCSF and XH is NH2 and is the N-terminal amine of the GCSF; L is a bond or a linking group; 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 by 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 C1-6 alkyl. ### Claim 5 (depends on 4) The compound according to claim 4 wherein L is a bond or is ### Claim 6 (depends on 1) A pharmaceutical composition comprising the compound of claim 1 and one or more pharmaceutically acceptable excipients. ### Claim 7 (depends on 1) A method for producing an amino terminal polysaccharide derivative of GCSF according to claim 1, which method comprises covalently coupling an anionic polysaccharide comprising 2-200 saccharide units attached to the amino-terminus of GCSF joined to one another through α2,8 or α2,9 linkages. ### Claim 8 (depends on 7) The method according to claim 7 wherein the anionic polysaccharide has a reactive aldehyde group which reacts with the GCSF and the coupling reaction is carried out under reducing conditions. ### Claim 9 (depends on 8) The method according to claim 8 wherein the reactive aldehyde group is at the non-reducing end of the anionic polysaccharide. ### Claim 10 (depends on 7) The method according to claim 7 wherein the polysaccharide reacts with an amine group of the GCSF. ### Claim 11 (depends on 10) The method according to claim 10 wherein the polysaccharide reacts with a terminal amine group of the GCSF protein. ### Claim 12 (depends on 10) The method accord to claim 10 wherein the amine is derived from a lysine amino acid side chain of the GCSF protein. ### Claim 13 (depends on 7) The method according to claim 7 wherein the anionic polysaccharide reacts with a terminal amine group of the GCSF in a first aqueous solution of pH of 4.0-6.0; and the resultant polysaccharide derivative is purified in a second aqueous solution of pH of 6.5-8.5. ### Claim 14 A compound which is a polysaccharide derivative of granulocyte colony-stimulating factor (GCSF) wherein the polysaccharide is anionic and comprises between 2 and 200 saccharide units. ### Claim 15 (depends on 14) The compound according to claim 14 wherein the polysaccharide is selected from polysialic acid, heparin, hyaluronic acid or chondroitin sulphate. ### Claim 16 (depends on 14) The compound according to claim 14 wherein the GCSF is derivatised by the polysaccharide at the reducing terminal unit of the polysaccharide. ### Claim 17 A compound of formula (I) XB is B—XH wherein B is GCSF and XH is NH2 and is an amine group of a lysine amino acid side chain of GCSF; L is a bond or a linking group; 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, heteroaryiene and alkylheteroarylene, any of which may substituted by 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 C1-6 alkyl. ### Claim 18 (depends on 17) The compound according to claim 17 wherein L is a bond or is ### Claim 19 (depends on 14) The pharmaceutical composition comprising the compound of claim 14 and one or more pharmaceutically acceptable excipients. ### Claim 20 (depends on 14) A method for producing a polysaccharide derivative of GCSF according to claim 14, which method comprises covalently coupling an anionic polysaccharide comprising 2-200 saccharide units attached to GCSF and joined to one another through α2,8 or α2,9 linkages. ### Claim 21 (depends on 20) The method according to claim 20 wherein the anionic polysaccharide has a reactive aldehyde group which reacts with the GCSF and the coupling reaction is carried out under reducing conditions. ### Claim 22 (depends on 21) The method according to claim 21 wherein the reactive aldehyde group is at the nonreducing end of the anionic polysaccharide. ### Claim 23 (depends on 20) The method according to claim 20 wherein the polysaccharide reacts with an amine group derived from a lysine amino acid side chain of the GCSF protein.