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Glycosaminoglycans

a technology of glycosaminoglycans and glycosaminoglycans, applied in the field of glycosaminoglycans, can solve the problems of unstable soluble peptide growth factor in physiological environments, unwanted side effects, and low yield

Inactive Publication Date: 2013-03-21
AGENCY FOR SCI TECH & RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a new polypeptide called heparan sulphate HS7, which can bind to a specific protein called SEQ ID NO:1. The invention also provides a method for obtaining and using HS7, as well as a pharmaceutical composition and a method of medical treatment using HS7. The technical effect of the invention is the discovery of a new molecule that can promote the growth of blood vessels, which can be used in the treatment of various diseases, conditions, or injuries to tissue.

Problems solved by technology

Due to its potent pro-angiogenic effects, VEGF has been trialled in clinical applications, but the outcomes have so far been disappointing 2,3,8.
The major obstacle is that this soluble peptide growth factor is unstable in physiological environments and rapidly degrades, and has to be administered at high (and excessive) dose that causes unwanted side effects.
It is also expensive to produce.
Whilst pro-angiogenesis therapies hold great promise for treating myocardial infarction, limb and other ischemic vascular diseases with high mortality, VEGF therapy for the treatment of ischemic disease has been questioned as therapeutic concentrations of soluble proteins are difficult to maintain at ischemic sites, and exogenous growth factors easily lose activity while being overdosed.
However, heparin is not suitable to stimulating angiogenesis in clinical applications.
Second, instead of selectively binding pro-angiogenic factors, heparin and mixtures of HS variants ubiquitously bind a variety of other soluble growth factors; given their inherent lack of specificity.
Some of these nonspecific bindings may bring forward unpredictable effects that may antagonise the fundamental process needed for controlled angiogenesis.

Method used

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Examples

Experimental program
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Effect test

example 1

Isolation of VEGF-Binding HS

[0183]We conjugated the biotinylated heparan-binding domain of VEGF165 (FIG. 1A) to a streptavidin column for isolating VEGF-affinity HS, so named HS7. A non-specific unbound HS(HSUB) fraction was eluted by low salt wash buffer, and only a single peak of bound affinity-trapped HS7 retrieved by high salt elution (FIG. 1B). We managed to purify ˜10 mg HS7 from 125 mg of commercially available porcine mucosal HS)(HSC). This proved the feasibility of acquiring a reasonable amount of VEGF-binding HS from an heterogeneous HS source.

example 2

Determination of HS7 Activity in Binding VEGF and Promoting Angiogenesis

[0184]We performed glycosaminoglycan enzyme-linked immunosorbant assay (GAG-ELISA) to evaluate the affinity of HS7 as compared with the crude HS material. As shown in FIG. 2A, HS7 binds significantly more VEGF165 (218%) than does HSC, proving its higher affinity to VEGF165. Next, we utilized the chick chorioallantoic membrane (CAM) assay to determine the possible pro-angiogenic activity of HS7 alone as compared with VEGF165 and saline controls. Both the sugar and the growth factor induced neovascularisation in the chicken embryo (FIG. 8), and HS7 exhibited moderately stronger pre-angiogenic efficacy compared with VEGF165, suggesting that HS7 alone could effectively stimulate blood vessel formation.

example 3

Purify and Fully Characterise VEGF165-Binding HS

[0185]We have expertise in isolating growth factor-affinitive HS species from different origins 16,19,22, and in our preliminary study the isolated HS7 (˜10% yield) has shown to be active. We are now seeking to fully characterise its composition and structure. All procedures generally follow our previously well established protocols 10,16,19,20,22-24.

Affinity Chromatography of VEGF165-binding HS

[0186]Purification of the VEGF-binding HS fractions is based on their affinity to the heparin-binding domain of VEGF165. Crude commercially available porcine mucosal heparan sulfate)(HSC) is purchased from Celsus Laboratories (Cincinnati, Ohio, USA), e.g. (e.g. INW-08-045, Heparan Sulphate I, Celsus Lab Inc, HO-03102, HO-10595, 10×100 mg.

[0187]A 55-amino acid peptide, ARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR [SEQ ID NO:1], corresponding to the heparin-binding domain of VEGF165, is synthesised and biotinylated by ITS Science and Me...

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Abstract

Heparan sulphate HS7 is disclosed, together with the use of HS7 in the growth and / or development and / or regeneration of tissue.

Description

PRIORITY CLAIM[0001]This application claims priority to Singapore Patent Application No. SG 201106706-3 filed Sep. 16, 2011, the entire contents of which are incorporated herein by reference.SEQUENCE LISTING[0002]In accordance with 37 CFR 1.52(e)(5), a Sequence Listing in the form of a text file (entitled “sequence_listing.txt,” created on Sep. 13, 2012, and 4 kilobytes in size) is incorporated herein by reference in its entirety.FIELD OF THE INVENTION[0003]The present invention relates to glycosaminoglycans capable of binding to VEGF, including their isolation and identification, and the use of the isolated glycosaminoglycans in the growth and / or development and / or regeneration of tissue.BACKGROUND TO THE INVENTION[0004]Ischemic heart and vascular diseases are the leading cause of death worldwide, accounting for more than 7 million deaths globally and 1 in 5 deaths in Singapore each year 1. Diseases, such as myocardial infarction, stroke and limb ischemia, occur due to blockage of ...

Claims

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Application Information

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IPC IPC(8): C08B37/00A61P9/00A61K9/00C12N5/071A61K38/18A61K31/737
CPCA61K31/737A61L27/34A61L27/54A61K38/1866A61L2300/412C08B37/0078C08L5/00A61K31/727A61P9/10
Inventor COOL, SIMONNURCOMBE, VICTOR
Owner AGENCY FOR SCI TECH & RES
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