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WO1994021689A1 - Oligosaccharides d'heparane-sulfate presentant une affinite de liaison avec le facteur de croissance des hepatocytes - Google Patents

Oligosaccharides d'heparane-sulfate presentant une affinite de liaison avec le facteur de croissance des hepatocytes Download PDF

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Publication number
WO1994021689A1
WO1994021689A1 PCT/GB1994/000615 GB9400615W WO9421689A1 WO 1994021689 A1 WO1994021689 A1 WO 1994021689A1 GB 9400615 W GB9400615 W GB 9400615W WO 9421689 A1 WO9421689 A1 WO 9421689A1
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Prior art keywords
oligosaccharide
hgf
chains
oligosaccharide preparation
idoa
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PCT/GB1994/000615
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English (en)
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Malcolm Lyon
John Thomas Gallagher
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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 AU62875/94A priority Critical patent/AU6287594A/en
Priority to EP94910470A priority patent/EP0642533A1/fr
Priority to JP6520835A priority patent/JPH07507596A/ja
Publication of WO1994021689A1 publication Critical patent/WO1994021689A1/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 certain novel oligosaccharide products and preparations thereof, useful in the field of biochemistry and medicine, which have particular binding affinity for certain growth factors or cytokines, in particular hepatocyte growth factor (HGF).
  • HGF hepatocyte growth factor
  • HGF hepatocyte growth factor
  • scatter factor hepatocyte growth factor
  • HGF is an unusually large (82kDa) and structurally complex growth factor that is synthesised as a biologically inactive single chain precursor. This is then proteolytically cleaved at a single site between linked cysteine residues giving rise to a disulphide-bonded heterodimer comprising a large ⁇ - chain ( 54kDa) containing a hairpin loop close to the N- terminus and a sequence of four Kringle domains, together with a smaller ⁇ -chain (26kDa).
  • HGF is produced by various cells including fibroblasts, smooth muscle cells, kidney mesangial cells and liver non-parenchymal cells. Its target cells are primarily epithelial cells, although it also acts on endothelial cells, hepatocytes and melanocytes. It is believed to play an important role as a paracrine mediator of epithelial-mesenchymal inter- actions. Cellular responses, however, to HGF are complex and, as well as being mitogenic, it can also stimulate cell migration and morphogenesis depending on the cellular target and its milieu. Interestingly, it can have an anti-proliferative effect on some tumour cells, including hepatoma cells, in vitro . It is likely that HGF is an important factor in embryonic organ developmen .
  • HGF heparan sulphate
  • HGF heparan sulphate
  • the present invention is based on studies in which we have shown that HGF does in fact interact in vitro with heparan sulphate. This has led to the isolation and at least partial characterisation of novel heparan sulphate oligosaccharides which exhibit significant binding affinity for HGF and which have certain structural features that contrast with those of other known growth factor binding oligosaccharides.
  • HGF hepatocyte growth factor
  • IdoA - ⁇ -L-iduronic acid (or iduronate); IdoA(2S) - ⁇ -L-iduronic acid 2-sulphate (or iduronate); GlcNAc - N-acetyl ⁇ -D-glucosamine; GlcNAc(6S) - N-acetyl ⁇ -D-glucosamine 6-sulphate; GICNSO3 - N-sulphated ⁇ -D-glucosamine; GlcNS ⁇ 3(6S)- N-sulphated ⁇ -D-glucosamine 6-sulphate; GlcNR - ⁇ -D-glucosamine with unspecified N- substituent; nUA - unsaturated uronic acid residue (e.g. DGlc for unsaturated D-glucuronic acid and DHex A for unsaturated unspecified hexuronic acid residue); SAX - strong-anion exchange; HPLC - high performance liquid
  • the invention provides novel oligosaccharides or preparations thereof which have a specific binding affinity for HGF.
  • Such oligosaccharides will generally be in the form of substantially homogenous preparations consisting of oligosaccharide chains composed of a sequence of at least three disaccharide units (dp>_6), preferably at least five disaccharide units (dp> ⁇ 10), and including a plurality of disaccharide units which each contain an IdoA( ⁇ 2S) and a GlcNS03( ⁇ 6S) residue and which preferably are arranged in between the terminal sugar residues of the oligosaccharide chains but not necessarily contiguously.
  • the oligo ⁇ saccharide chains will generally also be resistant to further depolymerisation by heparinase III (heparitinase - EC 4.2.2.8), and will be obtained from heparan sulphate or from other natural heparan type material.
  • heparinase III heparitinase - EC 4.2.2.8
  • the invention consists in an oligosaccharide preparation obtainable from partially depolymerised heparan sulphate (HS) or other natural heparin type material as a fraction thereof, characterised in that the oligosaccharide preparation consists essentially of oligosaccharide chains which have a specific binding affinity for hepatocyte growth factor (HGF) and which are composed of a sequence of at least three disaccharide units (dp ⁇ 6) that includes at least two disaccharide units containing an L-iduronic acid residue IdoA( ⁇ 2S) and an N-sulphated D-glucosamine residue GlcNS0 3 ( ⁇ 6S).
  • HGF hepatocyte growth factor
  • the invention can alternatively be defined as an oligosaccharide preparation comprising heparan sulphate (HS) fragments which have a specific binding affinity for hepatocyte growth factor (HGF) and which are composed of oligosaccharide chains containing a sequence of at least three disaccharide units (dp ⁇ 6) that includes at least two disaccharide units containing an L- iduronic acid residue IdoA( ⁇ 2S) and an N-sulphated D- glucosamine residue GlcNS03( ⁇ 6S) .
  • HGF hepatocyte growth factor
  • one or more of the above-mentioned at least two disaccharide units in the oligosaccharide chains is IdoA— ⁇ l,4—GlcNS03( 6S) , and the preparations may be such that the HGF-binding affinity is not completely destroyed by treatment under depolymerising conditions with heparinase I.
  • At least the majority of the oligosacc ⁇ haride chains may have substantially the same length as a result of carrying out a size fractionation separation procedure, and in preferred embodiments at least the majority of the oligosaccharide chains each have a degree of polymerisation (dp) of 10 or more, but with the maximum size being no greater than ten disaccharide units in total. More preferably, the oligosaccharide chains each have a degree of polymerisation (dp) of 12 or 14.
  • the oligosaccharide chains of the preparations of this invention are substantially completely resistant to further depolymerisation upon treatment under enzymic depolymerising conditions with heparinase III (heparit- inase I).
  • the IdoA(2S) content, if any, of said oligosaccharide chains will be less than the unsulphated IdoA content thereof, and in general the oligosaccharide chains will usually contain a relatively high proportion of 6—0—sulphated hexosamines compared to oligosaccharide chains of unmodified native heparan sulphate.
  • the content of glucosamine residues in the oligosaccharide chains which are O-sulphated at C6 will usually be greater than 24%, for example about 35% or greater. More specifically, the GlcNS03(6S) content of the oligosaccharide chains, i.e. number of residues per 100 disaccharides, is preferably at least 30% and may be 50% or more.
  • the structure of the oligosaccharide chains will include internal repeat sequences of IdoA( ⁇ 2S) and GlcNS ⁇ 3(6S) interrupted by occasional GlcNAc( ⁇ 6S) components, and in presently preferred embodiments substantially all said oligo ⁇ saccharide chains consist of a sequence of six or seven disaccharide units in all.
  • Oligosaccharide preparations of this invention will generally be obtainable by enzymic partial depolymerisa ⁇ tion to the fullest extent of heparan sulphate using heparinase III (heparitinase I), followed by size fractionation using, for example, gel filtration size exclusion chromatography, and then, in respect of a selected fraction or fractions recovered from the size fractionating stage, affinity chromatography using an HGF growth factor as the immobilised ligand in order to separate out HGF-binding fragments, and then eluting selectively over a range of salt concentrations under a salt gradient to fractionate said fragments in respect of HGF 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 of size fractionation and selection of recovered product.
  • the heparan sulphate (HS) may be derived from human fibroblast heparan sulphate proteoglycan (HSPG) or any other suitable biological
  • the invention may also be defined as providing an oligosaccharide preparation made up of oligosaccharide chains having a specific binding affinity for human hepatocyte growth factor (HGF), characterised in that
  • X is DHexA-GlcNS0 3
  • Y is IdoA( ⁇ 2S)-GlcR( ⁇ 6S),
  • Z is IdoA-GlcR where R is NSO3 or NAc, and n is in the range 1 to 5
  • n is three or more then at least for the majority of said molecular species two or more of the GICR3 residues in Y are N-sulphated glycosamines sulphated at C-6, i.e. GlcNS0 3 (6S); and (b) it is obtainable by a process comprising the steps of digesting a heparan sulphate with heparinase III (heparitinase I) so as to bring about partial depolymerisation thereof to the fullest extent, followed by size fractionating the oligosaccharide mixture produced using for example gel filtration size exclusion chromatography, collecting a fraction or fractions containing oligosaccharide chains having a particular size selected within the range of 10 to 20 monosaccharide residues, then subjecting said selected fraction or fractions to affinity chromatography using an immobilised HGF ligand and recovering the more strongly
  • HGF-binding constituents by eluting under a salt gradient over a range of salt concentrations and collecting a selected fraction or fractions containing the bound material which desorbs only at the highest salt concentrations.
  • the content of glucosamine residues having a 6-0-sulphate group will be greater than 24%.
  • the content of IdoA(2S), if any, will be small in these embodiments.
  • the invention also provides an oligosaccharide preparation having a specific binding affinity for hepato- cyte growth factors (HGF's) that is substantially wholly composed of oligosaccharide chains which are twelve or fourteen monosaccharide residues in length and which contain an internal sequence comprising at least 2 di ⁇ saccharide units each consisting of an IdoA residue linked to a GlcNS0 3 ( ⁇ 6S) residue, with more than 20% of the glucosamine residues (terminal or internal) being 6-0- sulphated.
  • HGF's hepato- cyte growth factors
  • substantially all the oligosaccharide chains may have the following sequence flGlcA-GlcNS0 3 -[IdoA-GlcNS0 3 ( ⁇ 6S)] n -IdoA-GlcR where R is NSO3 or NAc, and n is 4 or 5.
  • the present invention embraces a method or process for obtaining oligosaccharides that have a particular binding affinity for hepatocyte growth factor, characterised in that partial depolymerisation products of heparan sulphate, produced by treatment with a selective scission reagent that cleaves the polysaccharide chains thereof selectively in regions of relatively low sulphation, are subjected to affinity chromatography using HGF as the immobilised ligand so as to separate out HGF- binding fragments, the more strongly binding constituents then being recovered by eluting under a salt gradient and collecting a selected fraction or fractions containing the bound material which desorbs at the higher salt concen ⁇ trations.
  • the invention provides a method of isolating from heparan sulphate derived from heparan sulphate proteoglycan of mammalian cells low molecular weight oligosaccharides in a purified and relatively homogeneous state which have a specific binding affinity for hepatocyte growth factor, said method comprising the steps of (a) preparing an affinity chromatographic matrix or substrate incorporating a sample of hepatocyte growth factor (HGF) as the affinity ligand immobilised thereon;
  • HGF hepatocyte growth factor
  • step (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 five to seven in order to extract from the latter and retain on said matrix or substrate size selected oligo ⁇ saccharide fragments of the heparan sulphate glycosaminoglycan that have at least some binding affinity for the immobilised HGF;
  • step (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 selective scission reagent is heparinase III (heparitinase I) and the heparan sulphate is partially depolymerised to the fullest extent by digesting therewith until cleavage of the heparitase III sensitive linkages is complete.
  • the fractions collected from the size fractionation stage will preferably be those that appear to contain oligo ⁇ saccharides composed of six or seven disaccharide units.
  • the oligosaccharide preparations are applicable for therapeutic use, acting as an HGF-activity modulating agent for controlling or reducing cell growth, proliferation or migration in treating mammals in need of such treatment.
  • the invention also provides pharmaceutical formulations or compositions for medical use comprising a therapeutically effective non-toxic amount of an HGF-activity modulating agent comprising an oligosaccharide preparation as herein specified, or pharmaceutically acceptable salts thereof, together with a pharmaceutically acceptable carrier or vehicle.
  • a pharmaceutical composition or formulation in accordance with the invention for use in controlling the activity of hepatocyte growth factors in mammals may also be defined as comprising a therapeutically useful amount of an essentially pure oligosaccharide preparation having a specific binding affinity for hepatocyte growth factors (HGF's), consisting essentially of linear oligosaccharide chains which are substantially homogeneous with respect to HGF binding affinity and which contain a sequence of less than ten disaccharide units including, intermediate its terminal residues, a plurality of disaccharide units each composed of an N-sulphated glucosamine residue ( ⁇ 6S) and an unsulphated iduronic acid residue.
  • HGF's hepatocyte growth factors
  • FIGURE 1 This shows the comparative affinities of 3 H- heparin (panel A), 3 H 35 S-liver HSPG (panel B) and 3 H 35 S- liver HS chains (panel C) for an HGF affinity column, samples being applied in 0.15M NaCl and eluted with a step gradient of 0.2 - 1.0M NaCl as shown by the arrows in panel A.
  • FIGURE 2 This shows the effect of various specific modifications or depolymerisations of fibroblast HS on its affinity for HGF.
  • FIGURE 3 This shows a size fractionation of heparinase Ill-resistant oligosaccharides.
  • 3 H-fibroblast HS was exhaustively digested with heparinase III and the digest was fractionated into its constituent oligosaccharide sizes by gel filtration chromatography on Bio-Gel P10. Oligosaccharide fractions corresponding to dp2 - dpl2/14 (where dp is the number of monosaccharide units) were individually recovered.
  • FIGURE 4 This shows the effect of HS oligosaccharide size on HGF affinity.
  • 3 H-Fibroblast HS was digested with heparinase III and size fractionated on a Bio-Gel P10 column. Fractions corresponding to oligosaccharide sizes of dp6 (panel B), dp8 (panel C), dplO (panel D) and combined dpl2/14 (panel E) were tested for affinity to HGF and compared with the affinity of intact parent HS (panel A). Samples were applied in 0.15M NaCl and bound material was step eluted with increasing 0.2 - 1.0M NaCl concen- trations as shown by the arrows in panel A.
  • HS chains were prepared by exhaustive proteolytic digestion with Pronase.
  • HS chains were obtained from cultured foetal skin fibroblasts grown in MEM containing 10% (v/v) heat- inactivated donor calf serum (Gibco) and ImM glutamine. Confluent cultures were metabolically radiolabelled with lO ⁇ Ci/ml of D-[6- 3 H]-glucosamine hydrochloride for 72 hours.
  • the culture medium was removed and kept to one side whilst the cell layers were extracted with 0.15M NaCl, 20mM sodium phosphate, 1% (v/v) Triton X-100 pH 7.0 for 1 hour at room temperature with agitation.
  • the cell layer extracts were recombined with the culture supernatants and the whole was digested with Pronase ( lOO ⁇ g/ml ) for 3 hours at 37°C.
  • the digest was heated to 100°C for 5 minutes, clarified by centrifugation and then applied to a small DEAE-Sephacel column.
  • the intact HS chains were recovered by re-application of the digest to a small DEAE-Sephacel column, which was eluted as described above but omitting the Triton X-100.
  • the 3 H- HS chains were precipitated from the 1.5M NaCl eluant by addition of 3 vols of 95% (v/v) ethanol, air-dried and re- dissolved in distilled water.
  • the HS chains were selectively depolymerised either with heparinases or low pH nitrous acid, using methods performed essentially as described in Turnbull and Gallagher (Biochem, J. (1991), 273, 553-559). Solvolytic N-desulphation of fibroblast HS, followed by re-N- acetylation with acetic anhydride was also performed using the method of Inoue and Nagasawa (Carbohydr. Res. (1976), 46, 87-95). The content of the above-mentioned papers are incorporated herein by reference.
  • heparinase I (Flavobacterium heparinum; EC 4.2.2.7) referred to herein was supplied by Seikagaku Kogyo Co., Tokyo, Japan, but heparinase II (F. heparinum; no EC number assigned) and heparinase III (F. heparinium; EC 4.2.2.8) were from Grampian Enzymes of Aberdeen, Scotland.
  • Heparinase III is in fact substantially the same as the enzyme supplied under the designation heparitinase I by Seikagaku Kogyo Co.
  • Heparinase III (heparitinase I) will selectively 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). This is in contrast to the enzyme heparinase I (EC 4.2.2.7) which cleaves glycosidic linkages of disaccharides containing 2-sulphated L- iduronic acid.
  • heparinase I EC 4.2.2.7
  • HGF binding affinity of the HSPG, HS and oligosaccharide HS depolymerisation products was investigated using affinity chromatography with an HGF-affinity matrix or substrate, from which the HGF binding constituents were eluted and selectively recovered using a salt gradient.
  • Affi-Gel 10 (RTM) activated affinity gel from Bio-Rad Laboratories was washed following the supplier's instructions.
  • a portion of the recombinant human HGF (lOO ⁇ g) was pre-mixed with an excess of heparin (500 ⁇ g) in lOO ⁇ l of coupling buffer (0.1M HEPES, 80mM NaCl, pH 7.0) and incubated for 20 minutes at room temperature. The mixture was then added to 300 ⁇ l of the washed Affi-Gel 10 and the volume adjusted to 1ml with the coupling buffer.
  • the radiolabelled samples were each applied to the column and recirculated a number of times e.g. at a flow rate of 0.5ml/min. and at room temperature, so as to maximise opportunity to bind to the HGF.
  • the column was then washed with 5ml of 0.15M NaCl, 20mM sodium phosphate pH 7.0, followed sequentially with 5ml volumes of 0.2, 0.4, 0.6, 0.8 and 1.0M NaCl in 20mM sodium phosphate pH 7.0.
  • 0.1% (w/v) CHAPS was included in all the solutions. Fractions of 1ml were collected and monitored for radioactivity.
  • HS oligosaccharides 3 H fibroblast HS was degraded with heparinase III as described above. The digest was then separated into its constituent oligosaccharide size fractions by gel filtration chromatography on a Bio-Gel P10 column (1x115cm) eluted with 0.2M NH4HCO3 at a flow rate of 5ml/hr. The peaks corresponding to oligosaccharides from dp2 to a combined dpl2/14 fraction were individually pooled and repeatedly lyophilised to remove the NH4HCO3. Disaccharide composition of HS oligosaccharides
  • Disaccharide compositions of specific HS oligosaccharide fractions recovered from the affinity chromatography stage were analysed after exhaustive digestion and complete depolymerisation with a combination of the enzymes heparinase I, II and III.
  • the digestion mixture was generally made up of 20mIU/ml each of heparinases I, II and III in 0.1M sodium acetate, O.lmM calcium acetate, lmg bovine serum albumin/ml pH 7.0 at 37°C. Three additions of enzymes were made over an 18 hour digestion period.
  • the digest was then chromatographed on a Bio-Gel P2 column (1 x 111cm) eluted with 0.2M NH4HCO3 at a flow rate of 4ml/hr. Fractions corresponding to disaccharides were pooled, repeatedly lyophilised and finally re-dissolved in distilled water adjusted to pH 3.5 by the addition of HCl. Samples were then injected onto a Spherisorb (RTM) 5 ⁇ m SAX (strong anion-exchange) column (Technicol, Stockport, UK) linked to a Dionex HPLC system.
  • RTM Spherisorb
  • SAX strong anion-exchange
  • the column was washed with 5ml of acidified water pH 3.5 followed by elution of the constituent disaccharides with a 40ml gradient of 0-0.75M NaCl, pH 3.5 at a flow rate of lml/min.
  • the eluant was monitored with an on-line Radiomatic Flo-One/Beta Series A-200 radioactivity detector (Canberra Packard) using a 0.5ml flow-through liquid cell and a scintillant:sample ratio of 3:1.
  • the identities of the constituent disaccharides were determined by comparison with the elution positions of eight known disaccharide standards monitored by UV detection at 232nm.
  • liver HSPG bound strongly to the HGF affinity column with the majority of the bound material requiring 0.6 and 0.8M NaCl for eluting as shown in FIGURE IB.
  • the abundant unbound fraction would also bind if re-applied and was due to overloading of the column.
  • Pronase-released HS chains also bound strongly, although the proportion that eluted at the higher step (0.8M NaCl) was reduced (see FIGURE 1C). It is believed that the higher affinity of the intact HSPG may reflect the polyvalency of the HSPG and the greater possibility of bridging more than one immobilised HGF molecule.
  • N-sulphates per se make no more than a minor contribution to the binding activity and that the major binding determinants are other structural features spatially associated with the N-sulphation or GICNSO3 residues in such a way as to be similarly disrupted by nitrous acid depolymerisation treatment. Since both iduronate residues IdoA( ⁇ 2S) and 6-0-sulphated hexosamines are biosynthetically linked to the presence of N-sulphate, it was deduced that these are likely to provide the major binding determinants.
  • Heparinase I which specifically cleaves N- sulphated disaccharides containing IdoA(2S) residues, especially GlcNS03( ⁇ 6S)-IdoA(2S) , generated relatively large resistant fragments from fibroblast HS which would have internal sequences containing non-sulphated GlcA/IdoA.
  • the treatment had relatively little effect on HGF binding with most material eluting at 0.4M and 0.6M NaCl (see FIGURE 2E) .
  • HGF-binding fractions from a heparinase III digest of 3 H-fibroblast HS were analysed for their relative size distribution by gel filtration exclusion chromatography on Bio-Gel P10 (not shown). It was found that the non-binding (0.15M NaCl) and weakly bound (0.2M NaCl) fractions comprised predominantly dp2-4 oligosaccharides. In contrast, the medium (0.4M NaCl) and high (0.6M NaCl) affinity fractions contained oligosacch- arides of dp6-10 and dp> ⁇ 10 (mostly dpl2) respectively.
  • Size dependence was analysed in more depth by collecting individual oligosaccharide fractions from a preparative gel filtration chromatography fractionation of a large-scale heparinase III digest of 3 H-fibroblast HS.
  • the digest was fractionated on Bio-Gel P10 into oligo ⁇ saccharides ranging in size from dp2 to a mixed dpl2/14 fraction (see Fig. 3).
  • These oligosaccharide fractions were individually assayed for HGF-binding activity.
  • the general trend was for HGF affinity to increase with oligosaccharide size (see FIGURE 4). Dp2 and dp4 oligo ⁇ saccharides did not bind to HGF in 0.15M NaCl (data not shown).
  • oligosaccharides within the dpl0-12 size range probably comprise the smallest high affinity HGF-binding oligosaccharides.
  • the disaccharide compositions of dplO and dpl2/14 oligosaccharides with different binding affinities were further analysed to more positively identify structural features correlating with HGF affinity.
  • Oligosaccharides fractions recovered from the HGF column (Figs. 4D and 4E) were depolymerised by digesting using a combination of heparinases I, II and III and the resulting disaccharides were recovered by Bio-Gel P2 gel filtration chromato ⁇ graphy. The fractions were then analysed and identified using a SAX-HPLC column calibrated with known disaccharide standards.
  • N-sulphated disaccharides primarily flUA-GlcNS03( 6S) and nUA(2S)-GlcNS0 3 ( 6S) disaccharides
  • N-acetylated ( 6S) disaccharides whose abundance remained relatively constant.
  • These two N-sulphated( 6S) di ⁇ saccharides would be expected to contain IdoA or IdoA(2S) respectively in the original oligosaccharides and to be located internally.
  • the N-acetylated disaccharides would be expected to contain GlcA and could be derived from the reducing or non-reducing end (i.e. the sites of heparinase III cleavage), or might possibly be in an internal position where its environment may impart resistance to the enzyme.
  • Heparinase III digestion of HS will excise oligosaccharides which still retain most of the affinity for HGF.
  • the smallest such oligosaccharides found to have the high HGF affinity are decasaccharides (dplO), although the minimum binding sequence (minimum core sequence which retains useful high affinity) could perhaps be shorter than this.
  • Heparinase Ill-resistant sequences comprise mainly IdoA-containing disaccharides (with or without 2-sulphation) except for the non-reducing terminal, but non-sulphated IdoA residues appear to be important structural determinants of the HGF interaction.
  • GICNSO3 residues Clusters of IdoA(2S) residues are clearly not essential though there is some possibility that single residues may give some enhancement of binding.
  • Heparinase III- resistant oligosaccharides will contain GICNSO3 residues internally, but the results of the desulphation experiment indicate that these are not absolutely essential for the interaction and make only a modest contribution to the binding process.
  • GICNSO3 residues will, by necessity, be present in combination with IdoA( ⁇ 2S) residues as the latter can only be introduced into the polysaccharide adjacent to existing GICNSO3 residues.
  • oligo ⁇ saccharide preparations with a specific HGF-binding affinity have been obtained which are composed predom ⁇ inantly of oligosaccharide chains possessing one or more of the following features:
  • a GlcNS ⁇ 3(6S) content which is greater than 24%, for example about 30% or preferably greater, up to say about 50% or more.
  • HGF-binding oligo ⁇ saccharide products or preparations of the present invention the same basic techniques can be used as described above in connection with the background experi ⁇ mental work.
  • this can be partially depolymerised by treatment with heparinase III (or other equivalent selective scission reagent) and subjected to affinity chromatography using an HGF-affinity matrix or substrate and eluting under a salt gradient, then selectively collecting fractions eluting at the higher salt concent ⁇ rations to recover the material having the highest HGF- binding affinity, thereby providing a preparation of relatively short oligosaccharides which is substantially homogeneous with respect to HGF-binding affinity.
  • the oligosaccharides or preparations thereof in accordance with the invention can have a well defined composition and are readily capable of further purificat ⁇ ion if necessary, and considering also their relatively small sizes and specific HGF growth factor binding affinity, they can be very well suited for pharmaceutical use to exploit a potential in the field of medicine, e.g. as hepatocyte 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 HGF. This may arise for example where there is a need to control or modulate HGF-activity dependent cell growth and prolifer ⁇ ation or migration in clinical treatment of various conditions.
  • the oligosaccharide products may be made up into pharmaceutical formulations as required, and such uses are also within the scope of the invention.
  • 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 implicit ⁇ ly 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.
  • Heparinase Ill-resistant oligosaccharides of size dp lO and dpl2 14 were fractionated by affinity on HGF-Affigel. Oligosaccharide fractions eluted with

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  • Saccharide Compounds (AREA)

Abstract

Cette invention concerne des oligosaccharides qui présentent une forte affinité de liaison spécifique avec les facteurs de croissance des hépatocytes et qui sont constitués de chaînes d'oligosaccharides sulfatées de faible masse molaire. Les chaînes sont formées d'au moins trois unités disaccharides comprenant au moins une séquence interne constituée d'un reste N-sulfaté D-glucosamine 6-sulfate et d'un reste d'acide L-iduronique. Cette invention concerne également un procédé de préparation de ces oligosaccharides sous une forme purifiée et relativement homogène à partir d'héparane-sulfate. Pour que l'affinité de liaison avec le facteur de croissance des hépatocytes soit la meilleure possible, le nombre préféré d'unités disaccharides est au moins égal à cinq. Les structures les plus recherchées contiennent douze ou quatorze restes de monosaccharides au total et renferment une proportion relativement élevée d'hexosamines 6-0-sulfatées, c'est-à-dire plus de 30 % ou même 50 %, si on les compare aux chaînes d'oligosaccharides d'héparane-sulfate naturel non modifié. Ces oligosaccharides peuvent moduler l'activité du facteur de croissance des hépatocytes et peuvent être utilisées comme médicaments dans des applications thérapeutiques en médecine.
PCT/GB1994/000615 1993-03-25 1994-03-24 Oligosaccharides d'heparane-sulfate presentant une affinite de liaison avec le facteur de croissance des hepatocytes WO1994021689A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU62875/94A AU6287594A (en) 1993-03-25 1994-03-24 Heparan sulphate oligosaccharides having hepatocyte growth factor binding affinity
EP94910470A EP0642533A1 (fr) 1993-03-25 1994-03-24 Oligosaccharides d'heparane-sulfate presentant une affinite de liaison avec le facteur de croissance des hepatocytes
JP6520835A JPH07507596A (ja) 1993-03-25 1994-03-24 肝細胞成長因子結合親和性を有する硫酸ヘパラン・オリゴ糖類

Applications Claiming Priority (2)

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GB9306255.2 1993-03-25
GB939306255A GB9306255D0 (en) 1993-03-25 1993-03-25 Heparan sulphate oligosaccharides having hepatocyte growth factor binding affinity

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WO1994021689A1 true WO1994021689A1 (fr) 1994-09-29

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EP (1) EP0642533A1 (fr)
JP (1) JPH07507596A (fr)
AU (1) AU6287594A (fr)
CA (1) CA2136531A1 (fr)
GB (1) GB9306255D0 (fr)
WO (1) WO1994021689A1 (fr)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5620687A (en) * 1993-02-25 1997-04-15 Zymogenetics, Inc. Inhibition of intimal hyperplasia using antibodies to PDGF beta receptors
WO1998037222A1 (fr) * 1997-02-22 1998-08-27 Uhlenküken, Jochen Procede d'immobilisation reversible d'oligo et/ou de polysaccharides
US5976534A (en) * 1993-02-25 1999-11-02 Zymogenetics, Inc. Inhibition of intimal hyperplasia using antibodies to PDGF receptors and heparin
WO2000062785A1 (fr) * 1999-04-15 2000-10-26 Takara Shuzo Co., Ltd. Remedes
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
US6597996B1 (en) 1999-04-23 2003-07-22 Massachusetts Institute Of Technology Method for indentifying or characterizing properties of polymeric units
US6861254B1 (en) 1997-10-24 2005-03-01 Massachusetts Institute Of Technology Heparan sulfate D-glucosaminyl 3-O-sulfotransferases, and uses therefor
US7056504B1 (en) 1998-08-27 2006-06-06 Massachusetts Institute Of Technology Rationally designed heparinases derived from heparinase I and II
US7083937B2 (en) 2000-09-12 2006-08-01 Massachusetts Institute Of Technology Methods and products related to the analysis of polysaccarides
KR100727339B1 (ko) * 1999-04-15 2007-06-12 다카라 바이오 가부시키가이샤 치료제
KR100776956B1 (ko) 1999-04-15 2007-11-28 다카라 바이오 가부시키가이샤 치료제
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

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EP0014184A2 (fr) * 1979-01-08 1980-08-06 Kabi AB Fragments d'héparine ayant une activité anticoagulante sélective et procédé pour leur préparation
EP0244298A2 (fr) * 1986-04-17 1987-11-04 Sanofi Oligosaccharides hépariniques affinés pour les facteurs de croissance cellulaires
EP0509517A2 (fr) * 1991-04-16 1992-10-21 Seikagaku Kogyo Kabushiki Kaisha Oligosaccharide présentant une affinité pour le facteur de croissance des fibroblastes; procédé pour l'obtenir
EP0517182A1 (fr) * 1991-06-03 1992-12-09 Mitsubishi Chemical Corporation Agent à base de facteur de croissance d'hépatocyte
WO1993019096A1 (fr) * 1992-03-23 1993-09-30 Cancer Research Campaign Technology Limited Oligosaccharides presentant une affinite de liaison au facteur de croissance

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0014184A2 (fr) * 1979-01-08 1980-08-06 Kabi AB Fragments d'héparine ayant une activité anticoagulante sélective et procédé pour leur préparation
EP0244298A2 (fr) * 1986-04-17 1987-11-04 Sanofi Oligosaccharides hépariniques affinés pour les facteurs de croissance cellulaires
EP0509517A2 (fr) * 1991-04-16 1992-10-21 Seikagaku Kogyo Kabushiki Kaisha Oligosaccharide présentant une affinité pour le facteur de croissance des fibroblastes; procédé pour l'obtenir
EP0517182A1 (fr) * 1991-06-03 1992-12-09 Mitsubishi Chemical Corporation Agent à base de facteur de croissance d'hépatocyte
WO1993019096A1 (fr) * 1992-03-23 1993-09-30 Cancer Research Campaign Technology Limited Oligosaccharides presentant une affinite de liaison au facteur de croissance

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5976534A (en) * 1993-02-25 1999-11-02 Zymogenetics, Inc. Inhibition of intimal hyperplasia using antibodies to PDGF receptors and heparin
US5620687A (en) * 1993-02-25 1997-04-15 Zymogenetics, Inc. Inhibition of intimal hyperplasia using antibodies to PDGF beta receptors
US6217863B1 (en) 1995-10-30 2001-04-17 Massachusetts Institute Of Technology Rationally designed polysaccharide lyases derived from heparinase I
WO1998037222A1 (fr) * 1997-02-22 1998-08-27 Uhlenküken, Jochen Procede d'immobilisation reversible d'oligo et/ou de polysaccharides
EP0861903A1 (fr) * 1997-02-22 1998-09-02 Lansing, Manfred Procédé pour immobiliser de façon reversible des oligo- et polysaccharides
US6861254B1 (en) 1997-10-24 2005-03-01 Massachusetts Institute Of Technology Heparan sulfate D-glucosaminyl 3-O-sulfotransferases, and uses therefor
US7056504B1 (en) 1998-08-27 2006-06-06 Massachusetts Institute Of Technology Rationally designed heparinases derived from heparinase I and II
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
WO2000062785A1 (fr) * 1999-04-15 2000-10-26 Takara Shuzo Co., Ltd. Remedes
KR100776956B1 (ko) 1999-04-15 2007-11-28 다카라 바이오 가부시키가이샤 치료제
KR100727339B1 (ko) * 1999-04-15 2007-06-12 다카라 바이오 가부시키가이샤 치료제
US6597996B1 (en) 1999-04-23 2003-07-22 Massachusetts Institute Of Technology Method for indentifying or characterizing properties of polymeric units
US7412332B1 (en) 1999-04-23 2008-08-12 Massachusetts Institute Of Technology Method for analyzing polysaccharides
US7110889B2 (en) 1999-04-23 2006-09-19 Massachusetts Institute Of Technology Method for identifying or characterizing properties of polymeric units
US7117100B2 (en) 1999-04-23 2006-10-03 Massachusetts Institute Of Technology Method for the compositional analysis of polymers
US7139666B2 (en) 1999-04-23 2006-11-21 Massachusetts Institute Of Technology Method for identifying or characterizing properties of polymeric units
WO2001066772A3 (fr) * 2000-03-08 2002-05-02 Massachusetts Inst Technology Heparinase iii et ses utilisations
US7390633B2 (en) 2000-03-08 2008-06-24 Massachusetts Institute Of Technology Methods for preparing low molecular weight heparin with modified heparinase III
US7455986B2 (en) 2000-03-08 2008-11-25 Massachusetts Institute Of Technology, Inc. Heparinase III and methods of specifically cleaving therewith
US6869789B2 (en) 2000-03-08 2005-03-22 Massachusetts Institute Of Technology Heparinase III and uses thereof
US7939292B2 (en) 2000-03-08 2011-05-10 Massachusetts Institute Of Technology Modified heparinase III and methods of sequencing therewith
US7399604B2 (en) 2000-09-12 2008-07-15 Massachusetts Institute Of Technology Methods and products related to evaluating the quality of a polysaccharide
US7083937B2 (en) 2000-09-12 2006-08-01 Massachusetts Institute Of Technology Methods and products related to the analysis of polysaccarides
US7585642B2 (en) 2000-09-12 2009-09-08 Massachusetts Institute Of Technology Methods for evaluating the quality of a heparin sample
US7687479B2 (en) 2000-09-12 2010-03-30 Massachusetts Institute Of Technology Methods and producing low molecular weight heparin
US7709461B2 (en) 2000-10-18 2010-05-04 Massachusetts Institute Of Technology Methods and products related to pulmonary delivery of polysaccharides

Also Published As

Publication number Publication date
JPH07507596A (ja) 1995-08-24
EP0642533A1 (fr) 1995-03-15
AU6287594A (en) 1994-10-11
CA2136531A1 (fr) 1994-09-29
GB9306255D0 (en) 1993-05-19

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