Glycopegylated factor IX

a technology of glycopegylated factor and glycine, which is applied in the direction of drug compositions, peptide/protein ingredients, extracellular fluid disorder, etc., can solve the problems of inability to form blood clots, formation of unwanted blood clots, and most current therapies have undesirable side effects

Inactive Publication Date: 2006-02-23
NEOSE TECH
View PDF1 Cites 117 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0015] It has now been discovered that the controlled modification of Factor IX with one or more poly(ethylene glycol) moieties affords a novel Factor IX derivative with pharmacokinetic

Problems solved by technology

The regulation of blood coagulation is a process that presents a number of leading health problems, including both the failure to form blood clots as well as thrombosis, the formation of unwanted blood clots.
Unfortunately, most current therapies have undesirable side effects.
Warfarin therapy is complicated by the competitive nature of the drug with its target.
Fluctuations of dietary vitamin K can result in an over-dose or under-dose of Warfarin.
Fluctuations in coagulation activity are an undesirable outcome of this therapy.
A major inhibition to the use of vitamin K-dependent clotting factors is cost.
Overproduction of these proteins is limited by this enzyme system.
Furthermore, the effective dose of these proteins

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Glycopegylated factor IX
  • Glycopegylated factor IX
  • Glycopegylated factor IX

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0343] Preparation of Cysteine-PEG2 (2)

1.1 Synthesis of (1)

[0344] Potassium hydroxide (84.2 mg, 1.5 mmol, as a powder) was added to a solution of L-cysteine (93.7 mg, 0.75 mmol) in anhydrous methanol (20 mL) under argon. The mixture was stirred at room temperature for 30 min, and then mPEG-O-tosylate of molecular mass 20 kilodalton (Ts; 1.0 g, 0.05 mmol) was added in several portions over 2 hours. The mixture was stirred at room temperature for 5 days, and concentrated by rotary evaporation. The residue was diluted with water (30 mL), and stirred at room temperature for 2 hours to destroy any excess 20 kilodalton mPEG-O-tosylate. The solution was then neutralized with acetic acid, the pH adjusted to pH 5.0 and loaded onto a reverse phase chromatography (C-18 silica) column. The column was eluted with a gradient of methanol / water (the product elutes at about 70% methanol), product elution monitored by evaporative light scattering, and the appropriate fractions collected and dilut...

example 2

GlycoPEGylation of Factor IX Produced in CHO Cells

[0346] This example sets forth the preparation of asialoFactor IX and its sialylation with CMP-sialic acid-PEG.

2.1 Desialylation of rFactor IX

[0347] A recombinant form of Coagulation Factor IX (rFactor IX) was made in CHO cells. 6000 IU of rFactor IX were dissolved in a total of 12 mL USP H2O. This solution was transferred to a Centricon Plus 20, PL-10 centrifugal filter with another 6 mL USP H2O. The solution was concentrated to 2 mL and then diluted with 15 mL 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCl2, 0.05% NaN3 and then reconcentrated. The dilution / concentration was repeated 4 times to effectively change the buffer to a final volume of 3.0 mL. Of this solution, 2.9 mL (about 29 mg of rFactor IX) was transferred to a small plastic tube and to it was added 530 mU α2-3,6,8-Neuramimidase-agarose conjugate (Vibrio cholerae, Calbiochem, 450 μL). The reaction mixture was rotated gently for 26.5 hours at 32° C. The mixture was ...

example 3

Preparation of PEG (1 kDa and 10 kDa)-SA-Factor IX

[0348] Desialylated rFactor-IX (29 mg, 3 mL) was divided into two 1.5 mL (14.5 mg) samples in two 15 mL centrifuge tubes. Each solution was diluted with 12.67 mL 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 0.05% NaN3 and either CMP-SA-PEG-1 k or 10 k (7.25 μmol) was added. The tubes were inverted gently to mix and 2.9 U ST3Gal3 (326 μL) was added (total volume 14.5 mL). The tubes were inverted again and rotated gently for 65 hours at 32° C. The reactions were stopped by freezing at −20° C. 10 μg samples of the reactions were analyzed by SDS-PAGE. The PEGylated proteins were purified on a Toso Haas Biosep G3000SW (21.5×30 cm, 13 um) HPLC column with Dulbecco's Phosphate Buffered Saline, pH 7.1 (Gibco), 6 mL / min. The reaction and purification were monitored using SDS Page and IEF gels. Novex Tris-Glycine 4-20% 1 mm gels were loaded with 10 μL (10 μg) of samples after dilution with 2 μL of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% NaN3 buffe...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Volumeaaaaaaaaaa
Massaaaaaaaaaa
Massaaaaaaaaaa
Login to view more

Abstract

The present invention provides conjugates between Factor IX and PEG moieties. The conjugates are linked via an intact glycosyl linking group interposed between and covalently attached to the peptide and the modifying group. The conjugates are formed from glycosylated peptides by the action of a glycosyltransferase. The glycosyltransferase ligates a modified sugar moiety onto a glycosyl residue on the peptide. Also provided are methods for preparing the conjugates, methods for treating various disease conditions with the conjugates, and pharmaceutical formulations including the conjugates.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] The present application is a continuation-in-part of PCT Application No. PCT / US2004 / 041070, filed Dec. 3, 2004, and a non-provisional filing of U.S. Provisional Patent Application No. 60 / 684,729, filed May 25, 2005, each of which is incorporated herein by reference in its entirety for all purposes.BACKGROUND OF THE INVENTION [0002] Vitamin K-dependent proteins (e.g., Factor IX) contain 9 to 13 gamma-carboxyglutamic acid residues (Gla) in their amino terminal 45 residues. The Gla residues are produced by enzymes in the liver that utilize vitamin K to carboxylate the side chains of glutamic acid residues in protein precursors. Vitamin K-dependent proteins are involved in a number of biological processes, of which the best described is blood coagulation (reviewed in Nelsestuen, Vitam. Horm. 58: 355-389 (2000)). Vitamin K-dependent proteins include protein Z, protein S, prothrombin (Factor II), Factor X, Factor IX, protein C, Factor VII, G...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): A61K38/36
CPCA61K38/4846A61K47/48215A61K47/48092A61K47/549A61K47/60A61P7/00A61P7/04Y02A50/30
Inventor DEFREES, SHAWNBAYER, ROBERTBOWE, CARYNPANNEERSELVAM, KRISHNASAMY
Owner NEOSE TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap