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WO1993019096A1 - Oligosaccharides presentant une affinite de liaison au facteur de croissance - Google Patents

Oligosaccharides presentant une affinite de liaison au facteur de croissance Download PDF

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Publication number
WO1993019096A1
WO1993019096A1 PCT/GB1993/000597 GB9300597W WO9319096A1 WO 1993019096 A1 WO1993019096 A1 WO 1993019096A1 GB 9300597 W GB9300597 W GB 9300597W WO 9319096 A1 WO9319096 A1 WO 9319096A1
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Prior art keywords
oligosaccharide
fgf
sulphated
product
idoa
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PCT/GB1993/000597
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English (en)
Inventor
John Thomas Gallagher
Jeremy Ewan Turnbull
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Cancer Research Campaign Technology Limited
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Application filed by Cancer Research Campaign Technology Limited filed Critical Cancer Research Campaign Technology Limited
Priority to EP93906734A priority Critical patent/EP0632818A1/fr
Priority to JP5516393A priority patent/JPH07505179A/ja
Publication of WO1993019096A1 publication Critical patent/WO1993019096A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • C08B37/0078—Degradation products

Definitions

  • the present invention relates to the field of biochemistry and medicine. More particularly, it concerns certain novel oligosaccharide products and preparations thereof which have particular binding affinity for certain bioactive proteins or polypeptides present in biological systems, especially certain growth factors or cytokines such as fibroblast growth factors (FGF's). It also concerns uses of such oligosaccharide products, especially in medicine.
  • FGF's fibroblast growth factors
  • growth factors of which many have already been isolated and subsequently synthesised using recombinant DNA technology, are believed to act through a variety of mechanisms, but in general their effect appears to result from an initial interaction with specific receptors or binding sites on the surface of target cells which are thereby activated to bring about a chain or sequence of intracellular biochemical events.
  • Fibroblast growth factors characterised inter alia by a high binding affinity for heparin, are designated by the general term Fibroblast
  • FGF Fibroblast Growth Factor
  • aFGF acidic Fibroblast Growth Factor
  • bFGF basic Fibroblast Growth Factor
  • basic fibroblast growth factor appears to have an important role in processes such as embryonic development, wound repair and tumour growth, and it has been specifically implicated as being directly concerned in various disorders or degenerative conditions involving cell proliferation, including for example diabetic retinopathy, capsular opacification following cataract operations, restenosis after angioplasty, tumour angiogenesis, and various forms of chronic inflammation. It delivers its signal to cells by binding with specific cell surface tyrosine kinase receptors (K ⁇ 10-500 pM), such as receptors which are the expression products of the gene fig, that generate intracellular signals.
  • K ⁇ 10-500 pM specific cell surface tyrosine kinase receptors
  • HSPGs heparan sulphate proteoglycans
  • HS is probably the most complex mammalian glycosaminoglycan (GAG), consisting of a linear polysaccharide chain having an ordered arrangement of domains rich in N- and 0- sulphate groups, in which the basic disaccharide repeat unit consists of glucur ⁇ nic acid or iduronic acid linked to an N-sulphated glucosamine (i.e. GlcA/IdoA-GlcNS ⁇ 3), spaced apart by regions of low sulphation in which N-acetylated disaccharides (GlcA- GlcNAc) predominate.
  • GAG mammalian glycosaminoglycan
  • bFGF is a heparin-binding growth factor
  • the sulphated domains which contain some "heparin-like" regions might be expected to provide the most likely location of the bFGF binding site.
  • the size of these domains, their sulphation pattern and their iduronic acid content are highly variable, and a possibility arises that the strong interaction with bFGF may require a strictly defined sequence of sulphated monosaccharide isomers providing a specialised binding domain in a manner similar to the specific pentasaccharide sequence in heparin which has been found to have high affinity for antithrombin III.
  • Endothelial cell derived HS has already been demonstrated by affinity chromatography to bind strongly to bFGF, and a weaker interaction with HS from the Engelbreth Holm Swarm (EHS) tumour has also been reported, but the full structural requirements for such interactions have not previously been known.
  • EHS Engelbreth Holm Swarm
  • acidic fibroblast growth factor seems generally to be less potent, but nonetheless it is known as an active mitogen and differentiation factor for a wide variety of cells, especially mesodermal derived cell types, it is present in a variety of tissues, it binds to the same cell surface receptors as bFGF with substantially the same affinity, it likewise binds strongly to heparin and to the heparan sulphate of cell surface or extracellular matrix heparan sulphate proteoglycans, and the mechanism of interaction would appear to be the same as with bFGF.
  • aFGF acidic fibroblast growth factor
  • a number of other growth factors or cytokines also bind to heparan sulphate or similar sulphated glycosaminoglycans of extracellular matrix or cell surface proteoglycans, and again this may be a necessary prerequisite for their biological activity under physiological conditions.
  • Another possibility for blocking or reducing activity would be to employ agents that would act as antagonists or agonists to interfere with the preliminary binding interaction between such growth factors and the proteoglycan or glycosamino- glycan, such as heparan sulphate, which appears to be necessary before binding to the cell surface signal inducing receptors can take place, and for this purpose the possible use and administration of heparin for acting as a competitive inhibitor could be considered, at least in principle.
  • heparin or heparan sulphate itself
  • heparin is not particularly suitable for use as a drug in this context, not least because of its complexity and heterogeneity with a large number of different disaccharide sequences in its molecular composition such that it is likely to have multiple activities giving other undesirable effects and it would lack specificity.
  • What is needed for use as a drug is a high purity or substantially homogeneous preparation of a relatively small molecular compound of known composition which can be conveniently administered and which would have a very high degree of specificity for binding to the particular glycosaminoglycan binding sites of the growth factors in question with a low risk of promoting unpredictable or unwelcome side effects.
  • a molecule of minimal size consistent with high specific binding affinity, or a high value for the ratio of binding affinity or biological activity to size.
  • Such a drug could then provide a valuable regulatory therapeutic agent for blocking or inhibiting subsequent binding to the cell surface signal inducing receptors and thus reducing growth factor activity, or in other cases it might act to stimulate growth factor activity by promoting-subsequent growth factor binding to the cell surface signal inducing receptors.
  • growth factors at the time of administration may be complexed with a protective or activating agent in the form of a relatively small molecular compound as referred to above which could be co- administered with the growth factor and which would bind with a high degree of specificity to the glycosaminoglycan binding sites of the growth factor.
  • heparan sulphate can be partially depolymerised by selective scission reagents (e.g. enzymes such as heparinase and heparitinase) to yield preparations of relatively short length oligo ⁇ saccharides, such oligosaccharide preparations generally comprise a complex mixture of various molecular species having a wide range of different compositions and sizes.
  • selective scission reagents e.g. enzymes such as heparinase and heparitinase
  • the present invention has originated in the course of work which was undertaken to investigate human skin fibroblast heparan sulphate and which has led to the isolation and characterisation of distinct oligosaccharide structures having particular specific binding affinity for FGF's and similar heparin or heparan sulphate binding growth factors.
  • the invention enables oligosaccharide products to be prepared which, for medical use, especially as FGF growth factor modulating agents in connection with the treatment of various conditions herein referred to, are more suitable than any oligosaccharide preparations hitherto known.
  • GAG glycosaminoglycan
  • HS - heparan sulphate
  • HSPG heparan sulphate proteoglycan
  • bFGF basic fibroblast growth factor
  • aFGF - acidic fibroblast growth factor
  • dp degree of polymerisation
  • GlcA on the basis that it is believed to be derived from the saturated residue GlcA in an original polymer chain, e.g. based on the known specificity of heparitinase scission (see later); aMan R - 2,5-anhydro-D-mannitol formed by reduction of terminal 2,5-anhydromannose residues with NaBH 4 .
  • the symbol (n) is used to indicate that the monosaccharide residue concerned may or may not be unsaturated, and the symbol ( ⁇ 6S) denotes that a residue may or may not be sulphated at the C6 position.
  • the invention broadly provides novel oligosaccharide products having a high specific protein or polypeptide binding affinity, especially in respect of HS- binding proteins or polypeptides exemplified by growth factors such as FGF's.
  • the invention provides an oligosaccharide product having a specific binding affinity for fibroblast growth f ctors (FGF's), characterised in that it consists essentially of oligo ⁇ saccharide chains which are substantially homogeneous with respect to FGF binding affinity and-which contain at least four, preferably at least six, disaccharide units including sulphated disaccharide units, preferably arranged as a contiguous sequence, that are each composed of an N-sulphated glucosamine residue ( ⁇ 6S) and a 2-0- sulphated iduronic acid residue.
  • FGF's fibroblast growth f ctors
  • each of said sulphated disaccharide units is IdoA(2S)- ⁇ l,4-GlcNS03, and that the oligosaccharide chains consist of a sequence of less than ten disaccharide units in all.
  • the oligosaccharide chains may consist of a sequence of six disaccharide units in all of which at least four are included in the aforesaid contiguous sequence of sulphated disaccharide units, although in the most preferred embodiments there are a total of seven disaccharide units of which at least five are included in said contiguous sequence of sulphated disaccharide units.
  • the predominating majority of the oligosaccharide chains should all be of the same length and that the content (if any) of glucosamine residues O-sulphated at C6 should be less than 20%, or more preferably less than 5%.
  • Oligosaccharides in accordance with the invention will generally be substantially completely resistant to depolymerisation by heparitinase but not by heparinase, and may be obtainable from heparan sulphate (HS) of human fibroblast heparan sulphate proteoglycan (HSPG) by enzymic partial depolymerisation to the fullest extent with heparitinase followed by size fractionation, using for example gel filtration size exclusion chromatography, followed by, in respect of a selected fraction or fractions recovered from the size fractionating stage, affinity chromatography using an FGF growth factor as the immobilised ligand in order to separate out the FGF-binding fragments, and then eluting selectively over a range of salt concentrations under a salt gradient, advantageously a serially stepped gradient, to fractionate said fragments in respect of FGF binding affinity, followed by recovering the most strongly bound fragments and, optionally, further purifying the recovered product by carrying out at least one additional step
  • an oligosaccharide product having a specific binding affinity for fibroblast growth factors (FGF's) in accordance with the invention may be defined as being characterised in that
  • X is nHexA-GlcNS0 3 ( ⁇ 6S)
  • Y is IdoA(2S)-GlcNS0 3 ( ⁇ 6S
  • Z is IdoA-GlcR( ⁇ 6S) or IdoA(2S)-GlcR( ⁇ 6S) where R is NSO3 or NAc
  • n is in the range 4 to 7
  • Y is exclusively IdoA(2S)-GlcNS0 3 ,
  • n is 5 or 6 with there being a total of seven disaccharide units in all, or is 4 with there being a total of six disaccharide units in all, and
  • the invention may also be defined as providing an oligosaccharide product having a specific binding affinity for fibroblast growth factors (FGF's) that, at least in preferred embodiments, is substantially all composed of oligosaccharide chains which are either fourteen monosaccharide residues in length and which contain an internal contiguous sequence of 5 or 6 disaccharide units each consisting of an IdoA(2S) residue linked to a
  • FGF's fibroblast growth factors
  • GlcNS03( ⁇ 6S) residue with less than 20% of the glucosamine residues (terminal or internal) being 6-0- sulphated, or which are twelve monosaccharide residues in length and which contain an internal contiguous sequence of 4 disaccharide units each consisting of an IdoA(2S) residue linked to a GlcNS ⁇ 3( ⁇ 6S) residue, again with less than 20% of the glucosamine residues (terminal or internal) being 6-0-sulphated, the predominant oligo ⁇ saccharide chain sequence, accounting for substantially more than 50% of the component oligosaccharide chains and preferably more than at least 70% of the component oligo- saccharide chains, being preferably selected from the following:
  • Oligosaccharides in accordance with the invention include in particular the main constituent of the oligosaccharide product or preparation hereinafter designated oligo-H having a disaccharide sequence:
  • Oligosaccharides in accordance with the invention also include, however, related highly sulphated oligo ⁇ saccharides such as those comprising the main constituent of oligosaccharide preparations hereinafter designated oligo-M and oligo-L which have a weaker, but still significant, binding affinity to bFGF.
  • oligo-M oligosaccharide chains having the sequence nGlcA-GlcNS0 3 ( ⁇ 6S)-[IdoA(2S)-GICNSO3] 4 -Id ⁇ A-GlcR( ⁇ 6S) where R is generally NAc but may be NSO3
  • oligo-L appear to comprise the sequences nGlcA-GlcNS0 3 -Id ⁇ A-GlcNAc(6S)-GlcA-GlcNS0 3 (6S)- [Id ⁇ A(2S)-GlcNS0 3 ] 2 -IdoA-GlcR( ⁇ 6S) and nGlcA-GlcNS0 3 -IdoA(2S)-GlcNS0 3 -IdoA-GlcNAc(6S)-GlcA- GICNSO3(6S)- IdoA(2S)-GlcNS0 3 -Id ⁇ A-GlcR( ⁇ 6S) where R is generally NAc but may be NSO3
  • Oligosaccharide products in accordance with the invention may either be isolated from natural sources or may be made synthetically.
  • the invention further provides a method of isolating from a glycosaminoglycan such as heparan sulphate small oligosaccharides in a purified and relatively homogeneous state which have a specific binding affinity for a selected bioactive protein or polypeptide that itself binds to said glycosaminoglycan or to the corresponding proteoglycan in multicellular biological systems, said method comprising the steps of:
  • step (b) treating said glycosaminoglycan with a selective scission reagent so as to cleave the polysaccharide chains thereof selectively in regions of relatively low sulphation; (c) subjecting the product of step (b) to size fractionation, , for example by gel filtration size exclusion chromatography, and collecting selectively therefrom fractions that appear to contain oligosaccharides composed of less than ten disaccharide units,
  • step (d) contacting the affinity chromatographic matrix or substrate from step (a) with a selected fraction, or set of fractions, from step (c) containing a specific number of disaccharide units in the range of four to nine in order to extract from the latter and retain on said matrix or substrate size selected oligo- saccharide fragments of the glycosaminoglycan that have at least some binding affinity for the immobilised said protein or polypeptide;
  • step (f) collecting the fraction or set of fractions containing oligosaccharide fragments eluting in selected highest ranges of eluant salt concentration; and optionally, (g) further purifying the product of the selected fraction, or set of fractions, from step (f) by selectively repeating step (c) using said selected fraction or set of fractions collected in step (f) instead of the reaction mixture obtained from step (b), and optionally also repeating steps (d), (e) and (f).
  • the partial depolymerisation of the glycosaminoglycan may be carried out by a chemical method in which the polysaccharide is first N-deacetylated, e.g. by hydrazinolysis and is then treated with nitrous acid at about pH 4, this being used as the selective scission reagent, to bring about deaminitive cleavage at the free amino groups of the glucosamine residues resulting from the N-deacetylation.
  • the preferred selective scission reagent is the poly ⁇ saccharide lyase enzyme heparitinase which is commercially available from Seikagaku Corporation of Tokyo, Japan under the designation "Heparitinase I”, or from Sigma Chemical Co. under the designation "Heparinase III”, and which has the classification EC 4.2.2.8.
  • This enzyme will select- ively cleave glycosidic linkages on the non-reducing side of GlcA-containing disaccharides, such as in GlcNAc- ⁇ l,4- GlcA present in regions of low sulphation, but in general it will not cleave bonds of sulphated disaccharides containing L-iduronic acid or 2-sulphated L-iduronic acid, i.e. IdoA or IdoA(2S).
  • heparinase EC 4.2.2.7
  • heparinase cleaves glycosidic linkages between disaccharides containing 2-sulphated L- iduronic acid
  • heparitinase enzyme There are several known varieties of the heparitinase enzyme which have substantially the same linkage specificity but which vary for example in depolymerisation efficiency according to the size of the substrate molecules.
  • Heparitinase is used to denote the enzyme supplied by Seikagaku Corporation as “Heparitinase I”, or any other equivalent enzyme having the same glycosidic linkage specificity.
  • Oligosaccharides or oligosaccharide products in accordance with the invention generally have a well defined composit ⁇ ion, readily capable of further purification if necessary, and considering also their size and specific growth factor binding affinity they can be very well suited for pharmaceutical use to exploit a considerable potential in the field of medicine, e.g. as growth factor inhibitors or activators and mobilising agents. Accordingly, they are expected to have valuable applications as therapeutic drugs, particularly for controlling or regulating the activity of FGF's, especially bFGF.
  • FGF- activity dependent cell growth or proliferation in clinical treatment of conditions such as diabetic retinopathy, restenosis after angioplasty, capsular opacification, proliferation vitreoretinopathy, arthritis and other chronic inflammatory conditions, cancer cell growth and tumour angiogensis, mild muscular dystrophy, Alzheimer disease and various viral infections (e.g. Herpes Simplex type 1).
  • conditions such as diabetic retinopathy, restenosis after angioplasty, capsular opacification, proliferation vitreoretinopathy, arthritis and other chronic inflammatory conditions, cancer cell growth and tumour angiogensis, mild muscular dystrophy, Alzheimer disease and various viral infections (e.g. Herpes Simplex type 1).
  • oligosaccharide products may be made up into pharmaceutical formulations as required, and such uses are also within the scope of the invention.
  • FIGURE 1 This shows the fractionation of native and partially depolymerised HS on a bFGF-affinity column in experiments in which ⁇ H-labelled samples of HS chains (control - panel A), or HS treated with heparinase (panel B), or heparitinase (panel C), were fractionated on a bFGF affinity column as hereinafter described. Bound material was eluted with a step gradient of sodium chloride as shown in panel A (dotted line).
  • Heparitinase-resistant oligosaccharides retaining high affinity for bFGF were pooled, then dialysed (Spectrapor 7 1000-Mr cut ⁇ off, Spectrum UK) and freeze dried. Their size was then established by gel filtration on a Bio-Gel P6 column (1 x 120cm) at a flow rate of 4ml/hour in 0.5M NH 4 HC0 3 (panel D).
  • FIGURE 2 This shows the effect of heparinase on the affinity of HS oligosaccharides for bFGF in experiments in which 3 H-labelled HS chains were first treated with heparitinase and size fractionated by Bio-Gel P6 chromatography. Fractions of the heparitinase-resistant oligosaccharides of size dpl2 and dpl4 were pooled and then fractionated by bFGF affinity chromatography. Three major fractions eluting at 0.75M, 1.0M and >_ 1.25M NaCl were obtained, designated oligo-L (low), oligo-M (medium) and oligo-H (high) affinity oligosaccharides respectively.
  • FIGURE 3 This shows the results of Bio-Gel P6 chromatography of HS oligosaccharides having differing affinities for bFGF in experiments in which HS oligo ⁇ saccharides (dpl2-14) with relatively low (oligo-L), medium (oligo-M) and high (oligo-H) affinity for bFGF were prepared as in connection with Fig. 2.
  • Their size distribution was established by Bio-Gel P6 chromatography either intact (solid line) or after heparinase treatment (dashed line).
  • FIGURE 4 This shows the results of Bio-Gel P6 chromato ⁇ graphy of bFGF-binding HS oligosaccharides subjected to deaminitive scission in experiments in which HS oligo ⁇ saccharides (dp 12-14) with low (oligo-L), medium (oligo- M) and high (oligo-H) affinity for bFGF, prepared as described in connection with Fig. 2, were treated with nitrous acid and fractionated by Bio-Gel P6 chromato- graphy. The disaccharides (dp2) were partially resolved into mono-sulphated (main peak) and non-sulphated species.
  • FIGURE 5 Shows the results of Bio-Gel P6 chromatography of bFGF-binding oligosaccharides (Oligo-H and Oligo-M) after being subjected to heparitinase IV depolymerisation;
  • FIGURE 7 Shows graphs "A” and "B", for bFGF and aFGF respectively, illustrating the effect of size of HS- binding oligosaccharides and binding affinity in relation to growth factor activation;
  • FIGURE 8 Shows a typical result of Bio-Gel P6 gel filtration of a heparitinase digest of ⁇ H-labelled fibroblast HS prior to bFGF-affinity chromatography, as referred to in the Example described herein.
  • the human recombinant bFGF was prepared in a manner similar to that described previously for acidic FGF by Ke, Y. et al, (1990) Biochem Biophys. Res. Co ⁇ m. 171, 963-971. Briefly, the recombinant bFGF was purified by heparin- Sepharose chromatography and reverse phase or cation- exchange HPLC from lysates of bacterial cells, harbouring a PKK 233-2-bFGF construct (see Amann, E et al , (1985) Gene, 40, 183-190) encoding amino acids 1-155 of human bFGF (see Abraham, J.A.. et al , (1986) EMBO J. 5_, 2523- 2528), to yield a single compound of MW 17kDa on SDS-PAGE. The amino acid sequence was consistent with that of human bFGF and the recombinant protein possessed full biological activity.
  • HSPG and HS chains biosynthetically radiolabelled with ⁇ H-glucosamine were prepared from confluent cultures of adult human skin fibroblasts as described in a paper by Turnbull and Gallagher (see Turnbull, J.E. et al , (1991) Bioche ⁇ z. J. 273, 553-559), the content of which is incorporated herein by reference.
  • the work also involved the use of affinity chromatography and strong-anion exchange HPLC of disaccharides, the affinity chromatography involving a bFGF-Affi-Gel 10 affinity matrix.
  • affinity chromatography involving a bFGF-Affi-Gel 10 affinity matrix.
  • bFGF-Affi-Gel 10 R TM activated affinity gel from Bio-Rad Laboratories was washed four times with five volumes of double distilled water using centrifugation at 800g for 1 minute. Heparin (500 ⁇ g) was added to bFGF (500 ⁇ g in 3ml 0.6M NaCl, 25 mM Na 2 HP0 4 , pH 6.6) and mixed with lml of washed and packed Affi-Gel 10 overnight at 4 ⁇ C.
  • the Affinity chromatography was generally carried out as follows:
  • Disaccharides were recovered by Bio-Gel P2 R TM chromatography and separated by HPLC on a ProPac PA1 analytical column (4 x 250mm; Dionex, UK). After equilibration in mobile phase (double distilled water adjusted to pH 3.5 with HC1) at lml/minute samples were injected and disaccharides eluted with a linear gradient of sodium chloride (0 - 1M over 45 minutes) in the same mobile phase. The eluant was monitored in-line for UV absorbance (A232 for unlabelled disaccharides) and for radioactivity (Radiomatic Flo-one/Beta A-200 detector).
  • HSPG metabolically-labelled with ⁇ H-glucosamine
  • HS chains were prepared by Pronase treatment of the HSPG and applied to an affinity column prepared with human recombinant bFGF as hereinbefore described. Bound material was eluted stepwise with NaCl concentrations ranging from 0.25M - 2.0M in 0.25M steps. The majority of the HS bound strongly to bFGF, the major peak eluting at 1.25M NaCl (see Fig IA).
  • N-sulphate groups were confirmed by the findings that either deaminitive scission with nitrous acid, or N-desulphation/re-N-acetylation of HS, abolished the high affinity interaction (results not shown).
  • heparinase acts in the N-sulphated regions and specific- ally cleaves disaccharides that contain 2-O-sulphated iduronate i.e.
  • Heparinase scission of HS resulted in products with significantly reduced affinities for bFGF, elution occurring in the range 0.25 - 0.75M NaCl (see Fig. IB).
  • the effects of heparitinase digestion were even more marked with the majority of the material either failing to bind to the column or eluting at 0.25 - 0.75M NaCl (see Fig.lC).
  • a minor population of oligosaccharides in the heparitinase digest displayed an affinity for bFGF that was comparable to the intact HS (eluting in the range 1.0 to 1.5M NaCl).
  • oligo-L low
  • oligo-M medium
  • oligo-H high affinity oligo ⁇ saccharides.
  • Re-application of the fractions to the column confirmed their different affinities for bFGF (see Fig. 2).
  • Oligosaccharides of size dpl4 were mainly present in the oligo-H fraction whereas the oligo-M and oligo-L fractions were predominantly dpl2 (see Fig 3).
  • the disaccharide composition of the H, M and L oligosaccharides was assessed by polysaccharide lyase depolymerisation and strong anion exchange HPLC as hereinbefore described.
  • the calculated molar ratios are shown in Table 2.
  • the most striking aspect of the analyses was the high content of disulphated disaccharides of the type nHexA(2S)- ⁇ l,4-GlcNS0 3 , particularly in oligo-H and oligo-M (approximately 74% and 60% respectively of disaccharide units).
  • the heparinase sensitivity of these fractions Figs.
  • Oligo-H yielded 71% of labelled product eluting in the position of the standard IdoA(2S)- aMan R ; the remaining labelled product eluted as an unsulphated peak, corresponding to nGlcA-aMan R and IdoA- GlcNAc (results not shown).
  • Disaccharide composition of HS oligosaccharides with differing affinities for bFGF Disaccharide composition of HS oligosaccharides with differing affinities for bFGF
  • HS oligosaccharides (dpl2-14) with low (oligo-L), medium (oligo-M) and high (oligo-H) affinity for bFGF were prepared as described in connection with Fig. 2. Disaccharide composition was analysed by strong anion exchange HPLC as described.
  • This table shows the average relative molar ratios of the constituent disaccharides of the HS oligo ⁇ saccharides (based on the disaccharide composition data of Table 1) and the predominant average size of these oligo ⁇ saccharides.
  • heparitinase IV from Seikagaku Kogyo Co.
  • This enzyme has a similar linkage specificity to heparinase [i.e. GlcNS ⁇ 3( ⁇ 6S)- ⁇ (1-4)- IdoA(2S)] , but is much more efficient at cleaving small substrates (such as tetrasaccharides and hexasaccharides) which contain susceptible linkages.
  • oligo-H sulphated oligosaccharide fraction
  • fibroblast HS composed of a sequence of seven disaccharides bound particularly strongly to bFGF.
  • the dominant structural unit in the oligosaccharide was IdoA(2S)- ⁇ l,4-GlcNS ⁇ 3 (74% of disaccharides; Table 1) and both the 2-O-sulphate and the N-sulphate groups appeared to be essential for binding activity.
  • Analysis of the disaccharide composition following deaminitive scission confirmed that the identity of the uronic acid moiety of this disaccharide was IdoA(2S) and not GlcA(2S).
  • oligo-H was a product of heparitinase digestion it was deduced that the sequence of the principal or most predominant oligosaccharide component or components is:
  • oligo-M In the case of oligo-M, it has been established that the principal or predominant oligosaccharide chains have a sequence nGlcA-GlcNS0 3 ( ⁇ 6S)-[IdoA(2S)-GlcNS0 3 ] 4 -IdoA-GlcR( ⁇ 6S) where R is generally NAc but may be NSO3
  • oligo-H sequence identified here may not necessarily represent the minimal sequence for optimal binding to bFGF, but it seems noteworthy that full activation of bFGF (measured by its ability to bind to the fig receptor) requires heparin fragments of about the same size as oligo-H, i.e. dpl4-dpl6.
  • the related cytokine acidic FGF is also strongly activated by heparin oligosaccharides in this size range and has also been found to bind to oligo ⁇ saccharides of the kind herein identified.
  • oligo-H oligosaccharides of medium and low affinity for bFGF which, as oligosaccharide products resistant to heparitin ⁇ ase digestion, contain the same basic disaccharide repeat of IdoA-GlcNS ⁇ 3.
  • Oligo-H and oligo-M have similar degrees of sulphation (1.6 and 1.5 sulphates/disaccharide respectively) but oligo-M contains approximately 60% of disaccharides in the form of IdoA(2S)- ⁇ l,4-GlcNS ⁇ 3 compared to 74% in oligo-H.
  • oligo-M About 10% of amino sugars in oligo-M are 6-O-sulphated (Table 1) which in terms of overall sulphation largely offsets the lower concentrat ⁇ ions of IdoA(2S). The only other detectable difference between the two fractions is in size, oligo-M containing predominantly six disaccharides compared to seven in oligo-H (Fig. 3). It is believed that the combined effects of fragment size and enrichment of IdoA(2S) are the key properties that facilitate a stronger interaction of oligo-H with bFGF.
  • oligo-L size dpl2
  • oligo-L is still quite highly sulphated (1.3 sulphates/disaccharide) because of the higher content of GlcNS0 3 (6S) and GlcNAc(6S) (Table 1), and has some specific bFGF-binding activity so that the main oligosaccharide components thereof, having the sequences hereinbefore specified, may have some "utility.
  • the differential O-sulphation of the large N-sulphated oligosaccharides probably reflects a complex mechanism of HS biosynthesis in which the 2- and 6- sulphotransferases may be regulated independently.
  • a specific sequence consisting of GICNSO3 and IdoA(2S) appears to be designed for strong binding to bFGF, it is believed that sequences with different sulphation patterns, especially those with mixed 2- and 6-sulphate isomers, may interact with other HS- binding proteins and other members of the FGF family may bind preferentially with HS sequences which are slightly different to those preferentially recognized by bFGF.
  • glycosaminoglycans GAGs
  • the sequences of the antithrombin-III binding region in heparin was a major advance in this field (Lindahl, 1984). The interaction is specific, requiring a distinct sugar sequence and sulphat- ion pattern, rather than being determined mainly by relatively unspecific electrostatic forces.
  • Antithrombin- III is activated by heparin in a manner analogous to HS/heparin activation of bFGF.
  • This assay depended on the fact that 3T3 fibroblasts grown in the presence of the chemical sodium chlorate (which supresses polysaccharide sulphation) do not respond to aFGF or bFGF, but responsiveness (measured by incorpor ⁇ ation of 3 H-thymidine) is restored by addition of HS or heparin (as little as 1-lOng/ml) to the culture medium, thus allowing testing of the ability of exogenous HS oligosaccharides to activate FGFs.
  • oligo- saccharides with a range of structures and affinities for FGFs have been studied using this assay, in particular heparitinase-resistant oligos of size dp6, 8, 10, 12, 14 and 16 and larger (from porcine mucosal HS).
  • Preliminary results for both bFGF and aFGF are shown in Figure 7, and indicate that oligosaccharides dpl2 or larger are active, whereas those dplO or smaller are inactive.
  • the oligosaccharides of this invention may be conveniently prepared from purified heparan sulphate, native or recombinant, using the gel filtration chromatography and FGF-affinity chromatography techniques herein described in relation to the investigative experimental work, although generally the heparan sulphate may not need to be radiolabelled for purely preparative purposes. Oligosaccharides derived by heparitinase scission can readily be monitored by virtue of the unsaturated terminal uronic acid residue which absorbs strongly in the ultraviolet range (maximum at 232nm).
  • Confluent cultures of fibroblasts are maintained at 37 ⁇ C (C ⁇ 2/air, 1:19) in Eagle's minimal essential medium supplemented with 15% (v/v) donor-calf serum, 2mM-glut- amine, ImM-sodium pyruvate, non-essential amino acids, penicillin (100 units/ml) and streptomycin (lOO ⁇ g/ml).
  • Cells can be harvested at confluence, after biosynthetic radiolabelling if necessary [by incubating for 72 hours with Na 35 S0 4 (e.g. at 10-50 ⁇ Ci/ml) and/or [ 3 H]glucosamine (e.g. at 10-20 ⁇ Ci/ml)] .
  • HS can be extracted from both the medium and the cell layer. The medium is removed and the cell layers washed twice with warm (37 C C) phosphate-buffered saline (PBS). These combined solutions are centrifuged (200xg, 10 min) to pellet cells and other debris and the resulting supernatant constitutes the medium extract.
  • HS is efficiently extracted from the cell layers by treatment with 0.05% (w/v) trypsin in PBS at 37°C for 30 min.
  • the resulting cell suspension is centrifuged as above, and the supernatant removed carefully. After washing the pellet twice with PBS the combined supernatants constitute the cell layer trypsin extract.
  • the crude soluble extracts are subjected to initial purification by anion exchange chromatography.
  • Samples in PBS are loaded onto a DEAE-Sephacel column (1cm x 5cm) and washed with 0.3M NaCl in 20mM phosphate buffer, pH 6.8, to elute contaminating proteins and hyaluronic acid.
  • PGs and GAGs which remain bound are eluted with a gradient of 0.3- 1.0M NaCl in 20mM phosphate bufffer.
  • Fractions corresponding to HS are collected, pooled, desalted on a Sephadex G-25 column (2.5cm x 40cm) with distilled water as the eluant, and freeze dried.
  • Traces of contaminating GAGs e.g. chondroitin and dermatan sulphate
  • chrondroitinase ABC e.g. chondroitin and dermatan sulphate
  • Protein cores of HSPGs can then be removed by adding Pronase (5mg/ml final concentration) and calcium acetate (5mM final concentration) to the Chondroitinase ABC digest and digesting for 24 hours at 37°C.
  • HS chains are recovered by step elution from DEAE-Sephacel with 1M NaCl after eluting contaminants with 0.3 NaCl.
  • the fractions containing HS are then heated at 100°C for 10 minutes, followed by either dialysis against distilled water (using Spectrapor 7 high purity dialysis membrane) or by desalting on a Sephadex G-25 column as above, followed by freeze drying.
  • Biosynthetically 3 H and/or 35 S0 4 labelled HS chains purified as above are treated with heparitinase, i.e. heparitinase I (EC 4.2.2.8) from Seikagaku Kogyo Co, Tokyo, Japan, to provide cleavage within regions of relatively low sulphation while leaving intact the more highly sulphated domains rich in N- and O-sulphate groups and iduronate residues.
  • heparitinase i.e. heparitinase I (EC 4.2.2.8) from Seikagaku Kogyo Co, Tokyo, Japan
  • a sample of freeze dried HS (7xl0 6 dpm 3 H) is treated with heparitin ⁇ ase I (5 milli-units) in 200 ⁇ l of lOOmM Na acetate, pH 7.0, containing 0.2mM Ca acetate, at 37 ⁇ C, for 16 hours, followed by addition of a further aliquot of 5 milli-units of the heparitinase I and incubation for 1 hour at 37°C. Digestion is typically complete in 3-4 hours, but should normally be continued for 16 hours in order to ensure complete cleavage of all heparitinase-susceptible linkages.
  • An alternative chemical method for selective preparation of sulphated domains from HS is to specifically de-N-acetylate the polysaccharide, followed by specific cleavage at the resulting N-unsubstituted glucosamine residues.
  • the methodological details have been described in detail previously (Shaklee and Conrad (1984), Biochem. J. 217, 187-197; Guo and Conrad (1989), Analytical Biochemistr , 176, 96-104).
  • de-N- acetylation is carried out by hydrazinolysis by heating the sample at 96°C in 70% (w/v) aqueous hydrazine containing 1% (w/v) hydrazine sulphate, for approximately 4 hours.
  • oligo- saccharides which differ from those prepared by heparitin ⁇ ase treatment in that they terminate in intact hexuronate residues at their non-reducing ends and in 2,5-anhydro- mannose residues at their reducing ends.
  • Radiolabel can be introduced into the oligosaccharides at this stage, if required, by using NaB 3 H 4 as the reducing agent.
  • oligosaccharide products of the heparitinase (or chemical) treatment method are partially resolved on the basis of size by gel filtration chromatography, the result being individual peaks consisting of complex mixtures of oligosaccharides composed of defined numbers of di- saccharide units, ranging in size from disaccharides upwards, each differing from the next by an increase in size of one complete disaccharide unit.
  • analytical purposes e.g. sample loads up to approximately lOmg) columns (1 x 120cm or 1 x 240cm) packed with Bio-Gel P6 or Bio-Gel P10 (commercially available from Biorad Ltd.
  • Bio-Gel P10 is particularly suitable for separation of oligosaccharides larger than dplO in size.
  • the sample is loaded on to the top of the gel and eluted with 500mM NH4HCO3 at a flow rate of 4ml/hour. Fractions of lml are collected and a small aliquot taken from each for liquid scintillation counting if the HS has been labelled.
  • unlabelled HS oligosaccharides can be detected by measuring the absorbance at 232 nm, either of the individual fractions or continuously with a UV monitor.
  • Figure 8 shows a typical result for gel filtration of Bio-Gel P6 of the heparitinase digest of 3 H-labelled fibroblast HS described above.
  • bFGF-Affi-Gel 10 affinity matrix is packed into a glass column (bed dimensions 6mm x 35mm). Samples are loaded onto the column in lOmM Tris- HC1, pH 6.5, at a flow rate of 0.25 ml/min. Unbound material is eluted by collecting five lml fractions. Bound material is eluted with a gradient of sodium chloride (0-2M NaCl in column buffer) at a flow rate of 0.5 ml/min. This can be conveniently achieved by a discontinuous step gradient (e.g. increasing concentration of NaCl by steps of 250mM NaCl or other suitable increment). Five lml fractions are collected at each concentration.
  • a discontinuous step gradient e.g. increasing concentration of NaCl by steps of 250mM NaCl or other suitable increment.
  • a linear continuous gradient (e.g. with a total volume of 50ml) may be used to elute bound fragments, and lml fractions collected. A small aliquot is taken from each fraction for liquid scintillation counting (or detection by UV absorbance).
  • Figure 6 shows a typical result of the bFGF affinity chromatography of heparitinase-resistant oligosaccharides peaks of different sizes (dp2-dpl4) prepared by Bio-Gel P6 gel filtration.
  • Selected fractions containing oligo- saccharides having the same affinities for bFGF are pooled, desalted either by dialysis against distilled water using Spectrapor 7 1000-Mr cut-off membrane (Pierce Ltd) and/or by again using gel filtration on a Bio-Gel P2 column (1.5cm x 30cm) eluted with 500mM NH4HCO3 at a flow rate of lOml/hour, and freeze dried.
  • SAX strong anion exchange
  • PAGE gradient polyacrylamide gel electrophoresis
  • This methodology provides a very powerful technique for resolving complex mixtures of large oligosaccharides into single apparently homogeneous species, and it can be adapted to preparative scale for the separation of large quantities of oligosaccharides, either by eluting directly from the gel using appropriate apparatus or by electrotransfer from the gel onto a positively-charged nylon membrane, followed by recovery from the membrane by elution with salt as described in the above references.
  • precursors of these units designated B and which are to be arranged as B may be separately synthesised with O-acetyl or O-methylchloro- acetonyl (OMCA) protected terminal groups, e.g.
  • OMCA O-methylchloro- acetonyl
  • -0AC, IA OAC and MCAO—fcl The MCAO and OAC groups can then be converted selectively to —OH groups, e.g. by pyridine and hydrazine respectively, to enable firstly the required number of B units to be coupled together followed by the selective coupling of the required terminal units to build up the chain, and the structure produced can then be subjected to deacylation, O-sulphation, hydrogenolysis and N-sulphation as necessary to give the final product.
  • —OH groups e.g. by pyridine and hydrazine respectively
  • an effective growth factor binding amount of the active oligosaccharide which may be in the form of a pharmaceutically acceptable salt, will be made up as a pharmaceutical formulation ready for administration in any suitable manner, for example orally, parenterally (including subcutaneously, intramuscularly and intravenously), or topically, or in a slow-release dispensing device for implantation.
  • Such formulations may be presented in unit dosage form and may comprise a pharmaceutical composition, prepared by any of the methods well known in the art of pharmacy, in which the active oligosaccharide component or components is in intimate association or admixture with at least one other ingredient providing a compatible pharmaceutically acceptable carrier, diluent or excipient.
  • such formulations may comprise a protective envelope of compatible or relatively inert pharmaceutically acceptable material within which is contained the active oligosaccharide component or components with or without association or admixture with any other ingredients.
  • the oligosaccharides of the present invention may be in the form in which their non-reducing ends are unsaturated, as obtained by heparitinase scission, since there is some evidence that this form may be more resistant to bio-transformation which could reduce efficiency. However, this may not be essential for all applications.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active component, with capsules being a preferred type of formulation for providing the most effective means of oral delivery.
  • the formulations may comprise sterile liquid preparations of a predetermined amount of the active oligosaccharide component contained in sealed ampoules ready for use.
  • the amount of the oligosaccharide products of the invention, and dosing regimen required for effective therapeutic use will of course vary and will be ultimately at the discretion of the medical or veterinary practition ⁇ er treating the mammal in each particular case.
  • the factors to be considered by such a practitioner, e.g. a physician include not only the particular disorder being treated (and whether growth factor stimulation or growth factor inhibition is required) but also the route of administration and type of pharmaceutical formulation; the mammal's body weight; surface area, age and general condition.
  • a suitable effective bFGF inhibitory dose e.g.
  • the total daily dose may be given as a single dose, multiple doses, e.g. two to six times per day, or by intravenous infusion for any selected duration.
  • the dose range could perhaps be about 75 to 500 mg per day, and a typical dose would commonly be about 100 mg per day.
  • treatment might typically be 50mg of an oligosaccharide product, as hereinbefore defined, given 4 times per day in the form of a tablet, capsule, liquid (e.g. syrup) or injection.
  • the active oligo- saccharide component may be co-administered with the growth factor.
  • oligosaccharide compounds or products of the present invention will probably be most frequently targetted to the inhibition of growth factor activity, and pharmaceutical formulations or compositions containing these oligosaccharides are expected to be especially useful, as previously indicated, for treating conditions that arise, or are aggravated, as a result of activity of growth factors promoting harmful growth or cell proliferation, e.g.
  • diabetic retinopathy capsular opacification, proliferative vitreoretinopathy, tumour angiogenesis, cancer cell growth and metastasis, rheumatoid arthritis, mild muscular dystrophy, Alzheimer disease, various viral infections (e.g. Herpes Simplex type 1), or restenosis following angioplasty and other forms of chronic inflammation.
  • various viral infections e.g. Herpes Simplex type 1
  • restenosis following angioplasty e.g. Herpes Simplex type 1
  • the invention provides a number of different aspects and, in general, it embraces all novel and inventive features and aspects, including novel compounds, herein disclosed either explicitly or implicitly and either singly or in combination with one another. Moreover, the scope of the invention is not to be construed as being limited by the illustrative examples or by the terms and expressions used herein merely in a descriptive or explanatory sense.

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Abstract

On décrit des oligosaccharides présentant une affinité élevée de liaison spécifique envers les facteurs de croissance des fibroblastes (FCF) et composés de moins de dix unités disaccharides en tout. Ces oligosaccharides comprennent des unités disaccharides sulfatées composées d'un reste de glucosamine N-sulfaté et d'un reste d'acide iduronique 2-0-sulfaté. On décrit également un procédé de préparation de ces oligosaccharides dans un état purifié et relativement homogène à partir de glycosaminoglycanes tels que le sulfate d'héparane. Afin d'obtenir la meilleure affinité de liaison aux FCF, au moins quatre des unités disaccharides sulfatées sont disposées en séquence interne contiguë. Les meilleures structures contiennent quatorze restes monosaccharides en tout, mais des structures possédant douze restes monosaccharides peuvent également présenter une assez haute affinité de liaison aux FCF, au moins pour le facteur de croissance de fibroblaste basique (FCF-b). Ces oligosaccharides peuvent soit activer et stimuler soit inhiber l'activité des FCF. On décrit l'utilisation de ces oligosaccharides comme médicament à des fins thérapeutiques en médecine.
PCT/GB1993/000597 1992-03-23 1993-03-23 Oligosaccharides presentant une affinite de liaison au facteur de croissance WO1993019096A1 (fr)

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WO1994021689A1 (fr) * 1993-03-25 1994-09-29 Cancer Research Campaign Technology Limited Oligosaccharides d'heparane-sulfate presentant une affinite de liaison avec le facteur de croissance des hepatocytes
WO1996028730A1 (fr) * 1995-03-15 1996-09-19 Washington University Procede d'identification de molecules regulant l'activite du fgf
GB2300260A (en) * 1995-04-08 1996-10-30 Zeneca Ltd Determination of ligand binding affinity
WO1997039764A1 (fr) * 1996-04-23 1997-10-30 Toray Industries, Inc. Agent anti-pylori
US5696100A (en) * 1992-12-22 1997-12-09 Glycomed Incorporated Method for controlling O-desulfation of heparin and compositions produced thereby
US5766923A (en) * 1994-07-22 1998-06-16 President & Fellows Of Harvard College Isolated nucleic acid encoding ligands for FGFR
US5795875A (en) * 1994-05-06 1998-08-18 Glycomed Incorporated Therapeutic methods of using O-desulfated heparin derivatives
US5811403A (en) * 1996-09-30 1998-09-22 Vanderbilt University Polysaccharide toxin from Group B β-hemolytic Streptococcus (GBS) having improved purity
WO1999021588A1 (fr) * 1997-10-28 1999-05-06 Cancer Research Campaign Technology Limited Derives des facteurs de croissance des fibroblastes
US6217863B1 (en) 1995-10-30 2001-04-17 Massachusetts Institute Of Technology Rationally designed polysaccharide lyases derived from heparinase I
WO2001066772A3 (fr) * 2000-03-08 2002-05-02 Massachusetts Inst Technology Heparinase iii et ses utilisations
US6399386B1 (en) 1994-07-22 2002-06-04 President And Fellows Of Harvard College Method of isolating receptor and ligand DNA
US6597996B1 (en) 1999-04-23 2003-07-22 Massachusetts Institute Of Technology Method for indentifying or characterizing properties of polymeric units
EP1371666A2 (fr) * 1994-07-01 2003-12-17 Seikagaku Corporation Utilisation de l'héparine desulfatée
DE10258770B4 (de) * 2001-12-18 2005-02-10 F. Hoffmann-La Roche Ag Verfahren zum Testen eines Mittels auf dessen Fähigkeit, die Heparanaseaktivität zu hemmen
US6861254B1 (en) 1997-10-24 2005-03-01 Massachusetts Institute Of Technology Heparan sulfate D-glucosaminyl 3-O-sulfotransferases, and uses therefor
WO2005079817A1 (fr) * 2004-02-18 2005-09-01 The Texas A & M University System Heparine/heparane sulfate purifie a affinite destine a la regulation de l'activite biologique du recepteur fgf
US7056504B1 (en) 1998-08-27 2006-06-06 Massachusetts Institute Of Technology Rationally designed heparinases derived from heparinase I and II
US7081351B2 (en) 2001-12-18 2006-07-25 Hoffmann-La Roche Inc. Method for screening an agent for ability to inhibit heparanase activity
US7083937B2 (en) 2000-09-12 2006-08-01 Massachusetts Institute Of Technology Methods and products related to the analysis of polysaccarides
US7560106B2 (en) 1998-08-27 2009-07-14 Massachusetts Institute Of Technology Rationally designed heparinases derived from heparinase I and II and methods of sequencing therewith
US7709461B2 (en) 2000-10-18 2010-05-04 Massachusetts Institute Of Technology Methods and products related to pulmonary delivery of polysaccharides
WO2016080916A1 (fr) * 2014-11-19 2016-05-26 Agency For Science, Technology And Research Sulfates d'héparane à utiliser dans la réparation et/ou la régénération de la peau
WO2016111651A1 (fr) * 2015-01-09 2016-07-14 Agency For Science, Technology And Research Pdgf-b/pdgf-bb se liant à des variants de sulphates d'héparane
US10266612B2 (en) 2013-05-16 2019-04-23 Agency For Science, Technology And Research Heparan sulphates
WO2019117807A1 (fr) 2017-12-11 2019-06-20 Agency For Science, Technology And Research Oligosaccharides d'héparine et de sulfate d'héparane
US10493012B2 (en) 2014-11-19 2019-12-03 Agency For Science, Technology And Research Cosmetic use of heparan sulphate

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US7939292B2 (en) 2000-03-08 2011-05-10 Massachusetts Institute Of Technology Modified heparinase III and methods of sequencing therewith
US7390633B2 (en) 2000-03-08 2008-06-24 Massachusetts Institute Of Technology Methods for preparing low molecular weight heparin with modified heparinase III
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US10266612B2 (en) 2013-05-16 2019-04-23 Agency For Science, Technology And Research Heparan sulphates
WO2016080916A1 (fr) * 2014-11-19 2016-05-26 Agency For Science, Technology And Research Sulfates d'héparane à utiliser dans la réparation et/ou la régénération de la peau
US10471091B2 (en) 2014-11-19 2019-11-12 Agency For Science, Technology And Research Heparan sulphates for use in repair and/or regeneration of skin
US10493012B2 (en) 2014-11-19 2019-12-03 Agency For Science, Technology And Research Cosmetic use of heparan sulphate
WO2016111651A1 (fr) * 2015-01-09 2016-07-14 Agency For Science, Technology And Research Pdgf-b/pdgf-bb se liant à des variants de sulphates d'héparane
US10723813B2 (en) 2015-01-09 2020-07-28 Agency For Science, Technology And Research PDGF-B /PDGF-BB binding variants of heparan sulfates
WO2019117807A1 (fr) 2017-12-11 2019-06-20 Agency For Science, Technology And Research Oligosaccharides d'héparine et de sulfate d'héparane
US11331337B2 (en) 2017-12-11 2022-05-17 Agency For Science, Technology And Research Heparin and heparan sulphate oligosaccharides
US11806362B2 (en) 2017-12-11 2023-11-07 Agency For Science, Technology And Research Heparin and heparan sulphate oligosaccharides

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GB2265905A (en) 1993-10-13
CA2132750A1 (fr) 1993-09-30
JPH07505179A (ja) 1995-06-08
GB2265905B (en) 1995-01-25
GB9305979D0 (en) 1993-05-12
AU3763293A (en) 1993-10-21
GB9206291D0 (en) 1992-05-06
EP0632818A1 (fr) 1995-01-11

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