Polysaccharide-protein conjugates reversibly coupled via imine bonds

Abstract

A method of preparing an oxidised polysaccharide-protein conjugate by oxidising a polysaccharide with an oxidising agent to form an oxidised polysaccharide and combining such oxidised polysaccharide with a protein. The oxidised polysaccharide is reacted with a protein to form a composition comprising a conjugate wherein the oxidised polysaccharide and the protein are conjugated via one or more imine bonds and wherein the oxidised polysaccharide comprises essentially no alpha-hydroxy aldehyde units. The conjugate may be used to provide sustained or latent activity of the protein.

1 . A method of preparing an oxidised polysaccharide-protein composition comprising the steps of:
 (a) oxidising a polysaccharide with an oxidising agent to form an oxidised polysaccharide; and   (b) reacting the oxidised polysaccharide with a protein to form a composition comprising an oxidised polysaccharide-protein conjugate wherein the oxidised polysaccharide and the protein are conjugated via one or more imine bonds; and wherein the oxidised polysaccharide comprises essentially no alpha-hydroxy aldehyde units.   
     
     
         2 . A method according to  claim 1 , wherein the polysaccharide is selected from cellulose, pectin, starch and hydroxyhydrocarbyl derivatives thereof. 
     
     
         3 . A method according to  claim 1 , wherein the polysaccharide is selected from cellulose, pectin, starch, hydroxalkyl cellulose and hydroxalkyl starch. 
     
     
         4 . A method according to  claim 1 , wherein the polysaccharide is hydroxyethyl starch. 
     
     
         5 . A method according to  claim 1 , wherein the degree of oxidation of the oxidised polysaccharide is from 1 to 100%. 
     
     
         6 . A method according to  claim 1 , wherein the weight average molecular weight of the polysaccharide is from 1 to 2000 kDa. 
     
     
         7 . A method according to  claim 1 , wherein the ratio of oxidised polysaccharide to protein is from 0.1:1 to 20:1. 
     
     
         8 . A method according to  claim 1 , wherein the oxidised polysaccharide is reacted with a protein in step (b) in the presence of at least one further protein such that the oxidised polysaccharide-protein composition comprises more than one protein and each protein is conjugated to the oxidised polysaccharide via one or more imine bonds. 
     
     
         9 . A method according to  claim 1 , wherein the or each protein is selected from antibodies, cytokines, enzymes, growth factors and regulatory proteins. 
     
     
         10 . A method according to  claim 1 , wherein the or each protein is selected from erythropoietin (EPO), granulocyte-colony stimulating factor (G-CSF), uricase, beta-glucocerebrosidase, alpha-galactosidase, C-1 inhibitor, streptokinase, DNAseI, alpha-1 antitrypsin, asparaginase, arginine deiminase, Factor IX, Factor VIIa, Factor VIII, Factor IIa (thrombin), anti-TNF-alpha antibody, tissue plasminogen activator, human growth hormone, superoxide dismutase, catalase, CD163 antibody, anti-VEGF, anti-thrombin antibody, anti-CD20 antibody, anti-IgG1 antibody, anti-HER2 antibody, anti-CD33 antibody, anti-IgG2a antibody, anti-EGFR antibody, histone, interferon, insulin, albumin and mixtures thereof. 
     
     
         11 . A method according to  claim 1 , wherein the oxidised polysaccharide-protein conjugate is less than 0.1 μm in size. 
     
     
         12 . A method according to  claim 1 , wherein the protein has a weight average molecular weight of greater than about 1 kDa. 
     
     
         13 . A method according to  claim 1 , wherein the oxidised polysaccharide is reacted with a protein in step (b) in the absence of a molecular crowding agent. 
     
     
         14 . A method according to  claim 1 , wherein the oxidised polysaccharide is reacted with a protein in step (b) in the absence of a reducing agent. 
     
     
         15 . A method according to  claim 1 , wherein the oxidising agent is a periodate compound. 
     
     
         16 . A method according to  claim 1 , wherein the oxidised polysaccharide-protein composition further comprises non-conjugated protein. 
     
     
         17 . An oxidised polysaccharide-protein composition comprising an oxidised polysaccharide and a protein; wherein the oxidised polysaccharide comprises essentially no alpha-hydroxy aldehyde units and wherein the protein is conjugated to the oxidised polysaccharide via one or more imine bonds. 
     
     
         18 . An oxidised polysaccharide-protein composition according to  claim 17 , wherein the oxidised polysaccharide is derived from a polysaccharide selected from cellulose, pectin, starch and hydroxyhydrocarbyl derivatives thereof. 
     
     
         19 . An oxidised polysaccharide-protein composition according to  claim 17 , wherein the oxidised polysaccharide is derived from a polysaccharide selected from cellulose, pectin, starch, hydroxalkyl cellulose and hydroxalkyl starch. 
     
     
         20 . An oxidised polysaccharide-protein composition according to  claim 17 , wherein the oxidised polysaccharide is derived from hydroxylethyl starch. 
     
     
         21 . An oxidised polysaccharide-protein composition according to  claim 17 , wherein the degree of oxidation of the oxidised polysaccharide is from 1 to 100%. 
     
     
         22 . An oxidised polysaccharide-protein composition according to  claim 17 , wherein the oxidised polysaccharide is derived from a polysaccharide with a weight average molecular weight of the polysaccharide is from 1 to 2000 kDa. 
     
     
         23 . An oxidised polysaccharide-protein composition according to  claim 17 , wherein the ratio of oxidised polysaccharide to protein is from 0.1:1 to 20:1. 
     
     
         24 . An oxidised polysaccharide-protein composition according to  claim 17 , wherein the oxidised polysaccharide-protein composition further comprises at least one further protein and each protein is conjugated to the oxidised polysaccharide via one or more imine bonds. 
     
     
         25 . An oxidised polysaccharide-protein composition according to  claim 17 , wherein the or each protein is selected from antibodies, cytokines, enzymes, growth factors and regulatory proteins. 
     
     
         26 . An oxidised polysaccharide-protein composition according to  claim 17 , wherein the or each protein is selected from erythropoietin (EPO), granulocyte-colony stimulating factor (G-CSF), uricase, beta-glucocerebrosidase, alpha-galactosidase, C-1 inhibitor, streptokinase, DNAseI, alpha-1 antitrypsin, asparaginase, arginine deiminase, Factor IX, Factor VIIa, Factor VIII, Factor IIa (thrombin), anti-TNF-alpha antibody, tissue plasminogen activator, human growth hormone, superoxide dismutase, catalase, CD163 antibody, anti-VEGF, anti-thrombin antibody, anti-CD20 antibody, anti-IgG1 antibody, anti-HER2 antibody, anti-CD33 antibody, anti-IgG2a antibody, anti-EGFR antibody, histone, interferon, insulin, albumin and mixtures thereof. 
     
     
         27 . An oxidised polysaccharide-protein composition according to  claim 17 , wherein the oxidised polysaccharide-protein conjugate is less than 0.1 μm in size. 
     
     
         28 . An oxidised polysaccharide-protein composition according to  claim 17 , wherein the protein has a weight average molecular weight of greater than about 1 kDa. 
     
     
         29 . An oxidised polysaccharide-protein composition according to  claim 17 , wherein the oxidised polysaccharide-protein composition further comprises non-conjugated protein. 
     
     
         30 . An oxidised polysaccharide-protein composition according to  claim 17 , wherein the composition is obtained by the method as defined above. 
     
     
         31 . An oxidised polysaccharide-protein composition for use in the treatment or diagnosis of a disease or condition wherein the composition comprises an oxidised polysaccharide and a protein; wherein the oxidised polysaccharide comprises essentially no alpha-hydroxy aldehyde units and wherein the protein is conjugated to the oxidised polysaccharide via one or more imine bonds; and wherein the disease or condition is selected from hormone deficiency, homeostasis, thrombosis, metabolic enzyme deficiency, pulmonary disorder, gastrointestinal disorder, immunodeficiency, hematopoiesis, fertility disorders, immunoregulation, endocrine disorders, hemophilia, shock, growth regulation, cancer, transplantation, infectious disease, inflammatory and detoxification. 
     
     
         32 . An oixidised polysaccharide-protein composition according to  claim 31 , wherein the disease or condition is selected from hepatitis C virus (HCV) infection, acute lymphoblastic leukemia (ALL), chronic obstructive pulmonary disorder (COPD), alpha-1 antitrypsin (AAT) deficiency, anemia, chronic hyperuricemia, hemophilia, hemorrhage, chemotherapy-induced neutropenia, Gaucher's disease, Fabry's disease, hereditary angioedema, malignant melanoma, hepatocellular carcinoma (HCC), reperfusion injury, myocardial infraction, pulmonary embolism, psoriasis, Crohn's disease, rheumatoid arthritis, ulcerative colitis, cystic fibrosis, hemophilia A, hemophilia B, von Willebrand disease, diabetes, sepsis, hypovolemic shock and growth hormone deficiency. 
     
     
         33 . An oxidised polysaccharide-protein composition according to  claim 31 , wherein the composition is administered to a subject and the protein is released from the conjugate over time such that the circulatory half-life of the protein that is conjugated is enhanced relative to the circulatory half-life of non-conjugated protein. 
     
     
         34 . A method of reversibly conjugating a protein to an oxidised polysaccharide comprising the steps of preparing an oxidised polysaccharide-protein composition according to  claim 1 , and dissolving the oxidised polysaccharide-protein composition in a solvent. 
     
     
         35 . Use of an oxidised polysaccharide-protein composition according  claim 17  to solubilise the protein. 
     
     
         36 . Use according to  claim 35 , wherein the protein is insulin.