Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Stabilized alpha-galactosidase and uses thereof

A technology of galactosidase and galactose, applied in the field of polyprotein structure, can solve the problems of inability to start treatment, inability to provide solutions, inability to terminate, etc.

Active Publication Date: 2013-02-13
普罗塔里克斯有限公司
View PDF15 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These treatments only slow down the progression of the disease, but do not stop it, and do not provide a real and complete solution
Furthermore, in some cases ERT with commercial recombinant α-GAL had to be discontinued due to the development of an immunogenic response to treatment, and in some cases, treatment could not be started due to immunogenicity concerns

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
  • Stabilized alpha-galactosidase and uses thereof
  • Stabilized alpha-galactosidase and uses thereof
  • Stabilized alpha-galactosidase and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0395] Reference is now made to the following examples, which together with the foregoing description illustrate certain embodiments of the invention in a non-limiting manner.

[0396] Materials and methods

[0397] Material:

[0398] PEG from Iris Biotech GmbH 8 and 2000 Daltons (PEG 45 ) in PEG form, and from Pierce's PEG 5 Di-N-succinimide-poly(ethylene glycol) (di-NHS-PEG) was obtained in the form and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 25 mg / ml;

[0399] Citric acid was obtained from Sigma;

[0400]Coomassie Blue G250 from Bio-Rad;

[0401] Dimethylsulfoxide was obtained from Sigma;

[0402] D-(+)-galactose was obtained from Sigma;

[0403] Human plasma (K3 EDTA) was obtained from Bioreclamation Inc.;

[0404] 4-Methylumbelliferone was obtained from Sigma;

[0405] 4-Methylumbelliferone-A-D-galactopyranoside was obtained from Sigma;

[0406] N-Lauryl-nitrobenzoxadiazole-ceramide trihexoside (Gb 3 -NBD);

[0407] 2-(N-morphinyl)ethanesu...

Embodiment I

[0460] In Vitro Stability of Recombinant α-GAL

[0461] The in vitro stability of recombinant α-GAL was measured under various conditions as described above in the Materials and Methods section. testing of plant recombinant human α-GAL-I, and with Commercial recombinant human α-GAL.

[0462] Such as figure 1 As shown, all assay types of α-GAL showed loss of activity under simulated lysosomal conditions.

[0463] Additionally, if figure 2 As shown in , all assay types of α-GAL showed a loss of activity under simulated physiological conditions. As further shown there, under such conditions, the presence of 100 mg / ml galactose partially protected the activity of plant recombinant α-GAL-I.

[0464] Similarly, as in image 3 As shown in , in human plasma at 37°C, all tested types of α-GAL showed a loss of activity.

[0465] Such as Figure 4 It was shown that the presence of 100 mg / ml galactose partially protected the activity of plant recombinant α-GAL-I under simulate...

Embodiment II

[0469] Cross-linking of plant recombinant human α-GAL-I with di-N-hydroxysuccinimide-poly(ethylene glycol) (di-NHS-PEG) reagent

[0470] Plant recombinant human α-GAL-I (prh-α-GAL-I) was mixed with di-N-hydroxysuccinimide-poly (ethylene glycol) (di-NHS-PEG), that is, di-NHS-PEG 5 2-NHS-PEG 8 or Di-NHS-PEG 45 (di-NHS-PEG with 2000 Dalton PEG) cross-linked at Figure 5 Its structure is shown in .

[0471] Di-NHS-PEG can be attached to a protein at two sites on the protein, or at one site on the protein (eg, a lysine residue), thus forming a crosslink. exist Image 6 Both forms of connection are described in .

[0472] Add 100 μg of α-GAL-I in 28.5 μl of 2-(N-morphinyl)ethanesulfonic acid (MES) buffer (25 mM, pH 6) to 13.5 μl of phosphate buffer containing 100 mg / ml galactose (100mM, pH 8).

[0473] Di-NHS-PEG in 8 μl DMSO 5Add to α-GAL-I solution (1:50 molar ratio of 27.4 μg of α-GAL-I solution, 1:100 molar ratio of 54.8 μg of α-GAL-I solution, and 1:200 molar ratio of ...

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
molecular weightaaaaaaaaaa
molecular weightaaaaaaaaaa
Login to View More

Abstract

Multimeric protein structures comprising at least two alpha-galactosidase monomers being covalently linked to one another via a linking moiety are disclosed herein, as well a process for preparing same, and methods of treating Fabry disease via administration of a multimeric protein structure. The disclosed multimeric protein structures exhibit an improved performance, in terms of enhanced activity and / or a longer lasting activity under both lysosomal conditions and in a serum environment.

Description

technical field [0001] The present invention, in some of its embodiments, relates to novel multimeric protein structures (multimeric protein structures), more particularly, but not exclusively, to the multimeric protein structure of alpha-galactosidase and its role in the therapeutic Application in Fabry disease. Background technique [0002] During the catabolism of macromolecules, the lysosomal enzyme α-galactosidase-A (α-GAL or α-GalA; EC 3.2.1.22) catalyzes the removal of galactose from oligosaccharides, glycoproteins, and glycolipids. remove. Deficiencies of lysosomal enzymes lead to the accumulation of their substrates in tissues, a condition known as a lysosomal storage disease. In humans, deficiency of functional α-galactosidase-A leads to the accumulation in tissues of glycolipids (mainly glycosphingolipids (globotriaosylceramide) containing terminal α-galactose residues ), which is also known as "ceramide trihexoside" (ceramide trihexoside), "CTH" or "Gb 3 ”), ...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/40
CPCC12N9/2465C12Y302/01022A61P1/16A61P13/12A61P17/00A61P27/02A61P3/00A61P43/00A61P9/00A61P17/02A61P19/02A61P29/00A61P35/00C07K14/525A61K38/47
Inventor 阿维多尔·舒尔曼伊利亚·鲁德费尔泰里拉·本-摩西塔利亚·谢赫提尔亚尼瓦·阿聚莱约瑟夫·沙尔提埃尔塔利·基日涅尔
Owner 普罗塔里克斯有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products