WO1996023003A1 - Molecule therapeutique - Google Patents

Molecule therapeutique Download PDF

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Publication number
WO1996023003A1
WO1996023003A1 PCT/AU1996/000034 AU9600034W WO9623003A1 WO 1996023003 A1 WO1996023003 A1 WO 1996023003A1 AU 9600034 W AU9600034 W AU 9600034W WO 9623003 A1 WO9623003 A1 WO 9623003A1
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WO
WIPO (PCT)
Prior art keywords
fgf
cells
gag
derivative
hspg
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PCT/AU1996/000034
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English (en)
Inventor
Victor Nurcombe
Perry Francis Bartlett
Original Assignee
Amrad Operations Pty. Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AUPN0784A external-priority patent/AUPN078495A0/en
Priority claimed from AUPN3560A external-priority patent/AUPN356095A0/en
Application filed by Amrad Operations Pty. Ltd. filed Critical Amrad Operations Pty. Ltd.
Priority to AU44753/96A priority Critical patent/AU4475396A/en
Publication of WO1996023003A1 publication Critical patent/WO1996023003A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • 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

Definitions

  • the present invention is directed generally to a therapeutic molecule. More particularly, the present invention provides a therapeutic molecule comprising a heparan sulfate polymer or its derivative obtainable from heparan sulfate proteoglycan and which is capable of interacting with a specific cytokine.
  • the therapeutic molecule of the present invention will be useful in promoting cytokine function in vitro and in vivo.
  • the present invention is particularly directed to the effects of the heparan sulfate polymers on Fibroblast Growth Factors.
  • Fibroblast Growth Factor- 1 also known as acidic (a) FGF
  • Fibroblast Growth Factor-2 also known as basic (b) FGF
  • FGF-1 Fibroblast Growth Factor-1
  • FGF-2 Fibroblast Growth Factor-2
  • la;b heparin analogues
  • HSPG heparan sulfate proteoglycan
  • the HSPGs are a highly diverse group of macromoiecules, each of which consists of a core protein to which highly sulfated glycosaminoglycan (GAG) side chains of heparan sulfate are covalently attached (3a;b). They are ubiquitous constituents of mammalian cell surfaces and of most extracellular matrices including the specialised basal laminae that surround neural tissue (2a;b;4).
  • GAG glycosaminoglycan
  • the GAG side chains or various derivatives thereof on HSPGs exhibit specificity for particular cytokines such as FGF-1 or FGF-2.
  • a common core protein is synthesised with a particular species of polymeric GAG side chains which exhibit specificity, for example, to FGF-1 or FGF-2.
  • This discovery will result in a new generation of therapeutic molecules capable of modulating cytokine-ligand interaction and more particularly FGF-ligand interaction.
  • the present invention provides a range of derivatives and more specifically functional fragments of the GAG side chains which are especially useful in generating a range of therapeutic molecules.
  • one aspect of the present invention is directed to an isolated GAG polymer or derivative thereof from an HSPG wherein said GAG polymer or its derivative is capable of interaction with a cytokine. More particularly, the present invention provides an isolated GAG polymer or a derivative thereof from an HSPG wherein said GAG polymer or its derivative is capable of interaction separately with either FGF-1 or FGF-2 but not both. Even more particularly, the present invention contemplates an isolated GAG polymer or a derivative thereof from an HSPG wherein said GAG polymer or its derivative is capable of interaction with FGF-2 and wherein said HSPG is obtainable from murine cells at approximately embryonic day 8-10.
  • the present invention relates to an isolated GAG polymer or a derivative thereof from an HSPG wherein said GAG polymer or its derivative is capable of interaction with FGF-1 and wherein said HSPG is obtainable from murine cells at approximately embryonic day 11 or older such as day 11-13.
  • the murine cells are embryonic day 10 neuroepithelial cells transformed with an oncogene on a retroviral vector.
  • An example of a suitable cell line is the 2.3D cell line which is FGF sensitive (la;b).
  • the 2.3D cell line was deposited at the PHLS Centre for Applied Microbiology and Research, European Collection of Animal Cell Cultures (ECACC), Division of Biologies, Porton Down, Salisbury, Wiltshire SP4 0JG on 16 May, 1995 under Provisional Accession Number 95061001; the confirmed Accession Number is 95051601.
  • a particularly preferred embodiment of the subject invention is directed to an isolated GAG polymer or a derivative thereof from an HSPG wherein said GAG polymer or its derivative is capable of interaction with FGF-2 but not FGF-1 and wherein said HSPG is obtainable from the 2.3D cell line dividing freely in tissue culture.
  • This GAG polymer is referred to herein as "GAGB”.
  • a fragment or derivative includes molecules capable of promoting FGF binding to its receptor or inhibiting binding to its receptor. The derivatives may act, therefore, as agonists or antagonists of FGF binding to its receptor or of FGF-HSPG interaction.
  • the present invention is directed to an isolated GAG polymer or a derivative thereof from an HSPG wherein said GAG polymer or its derivative is capable of interaction with FGF-1 but not FGF-2 and wherein said HSPG is obtainable from the 2.3D cell line grown in culture under contact inhibiting conditions.
  • GAG polymer is referred to herein as "GAGA”.
  • polymer or like derivations includes molecules comprising at least four sugars or derivatives thereof such as from four to 400. Accordingly, another aspect of the present invention provides an isolated molecule comprising: (i) a repeating disaccharide structure (X-Y) n wherein: X is hexuronic acid; Y is glucosamine; and n is 2 to 200; (ii) an ability to bind either FGF-1 or FGF-2 but not both; and (iii) being isolatable from an HSPG which in one form comprises GAG polymers capable of binding FGF-1 and in another form comprises GAG polymers capable of binding FGF-2.
  • X-Y repeating disaccharide structure
  • X-Y are ⁇ , ⁇ -linked glucosamine and hexuronic acid in linkage sequence [(l ⁇ 4) ⁇ -D-glucosaminyl-(l ⁇ 4) ⁇ -D-hexuronosyl] n .
  • the glucosamine may be N- acetylated or N-sulfated and the hexuronate may be glucuronate or iduronate.
  • n is 2 to 20. More preferably, n is 4 to 15. Even more preferably n is 8 to 12 such as 9.
  • the specificity of a particular GAG polymer for a particular cytokine is created by the specific pattern of carboxyl and sulfate groups attached to the glucosamine and hexuronic acid.
  • a GAG polymer having the identifying characteristics of GAGB and comprising the following disaccharides in percentage amounts given in parentheses: iduronic acid N-acetylated glucosamine [UA-GlcNAc](55.4%), iduronic acid N-sulfated glucosamine [UA-GlcNSO 3 ] (22.2%), iduronic acid N-acetylated glucosamine 6-sulfate [UA-GlcNAc( ⁇ S)] (3.2%), iduronic acid 2-sulfate N-acetylated glucosamine [UA-(2S GlcNAc] (1.8%), iduronic acid N-sulfated glucosamine 6-sulfate [UA-GlcNSO 3 (6S)] (2.5%), iduromc acid 2-sulfate N-sulfated glucosamine [UA-(2S)-Gl
  • a GAG polymer having the identifying characteristics of GAGA and comprising the following disaccharides in percentage amounts given in parentheses: iduronic acid N-acetylated glucosamine [UA-GlcNAc] (50.7%), iduromc acid N-sulfated glucosamine [UA-GlcNSO 3 ] (19.1%), iduronic acid N-acetylated glucosamine 6-sulfate [UA-GlcNAc (6S)] (4.7%), iduronic acid 2-sulfate N-acetylated glucosamine [UA-(2S)- GlcNAc] (2.6%), iduronic acid N-sulfated glucosamine 6-sulfate [UA-GlcNSO 3 (6S)] (2.8%), iduronic acid 2-sulfate N-sulfated glucosamine [UA-(2S)-GlcNSO 3
  • the identifying characteristics of GAGB or GAGA include the preferential interaction with FGF2 and FGF1, respectively.
  • isolated is meant a preparation of a GAG polymer or a derivative thereof which has undergone at least one purification or separation step away from a core protein.
  • the term “isolated” extends to a biologically pure preparation of the polymer comprising at least 35%, preferably at least 45%, more preferably at least 55%, still more preferably at least 65%, even more preferably at least 75-80% and even more preferably greater than 95% of the GAG polymer as determined by weight, activity (e.g. cytokine binding activity), immunoreactivity (e.g. antibody interactivity), sugar content or other convenient means.
  • the GAG polymer or derivative thereof is purified from HSPG derived from conditioned medium produced by either the neuroepithelial cell line 2.3D which expresses the c-myc oncogene in cloned embryonic primary neuroepithelial cells or from primary neuroepithelial brain.
  • HSPG derived from conditioned medium produced by either the neuroepithelial cell line 2.3D which expresses the c-myc oncogene in cloned embryonic primary neuroepithelial cells or from primary neuroepithelial brain.
  • both neuroepithelial tissue at embryonic day 9 referred to herein as 9" or 2.3 D cells grown in non-confluent culture produces HSPG capable of binding FGF-2.
  • Embryonic day 11 tissue referred to herein as "El l”
  • 2.3 D cells grown in continuously confluent culture [13] i.e. under contact inhibiting conditions
  • the present invention is predicated in part on the discovery that the heparan sulfate side chains on 2.3 D non-confluent cells or E9 HSPGs bind FGF-2 while the heparan sulfate side chains on 2.3 D continuously confluent cells or El 1 HSPGs bind FGF-1.
  • the 2.3 D cell line provides, therefore, a particularly useful source of HSPG side chains and which can be readily upgraded to large scale commercial production.
  • another aspect of the present invention contemplates a method of purifying a GAG polymer or a derivative thereof capable of binding either FGF-1 or FGF-2, said method comprising generating a neuroepithelial cell line expressing an oncogene and growing and/or maintaining the cell line for a time and under conditions sufficient for said cell line to secrete HSPG molecules into the conditioned medium; collecting the HSPG at predetermined time intervals and subjecting same to HSPG isolating means; subjecting isolated HSPG to GAG polymer purification means.
  • a suitable protocol for purifying GAG polymers away from HSPG includes but is not limited to subjecting the HSPG to one or more proteolytic enzymes to destroy or substantially remove the protein core. Pronase is a particularly useful enzyme in this respect. Alternatively, the protein core may be removed by sonic disruption, shearing or via various forms of hydrolysis. HPLC or other suitable means may then be used to purify the GAG polymers.
  • the neuroepithelial cell line is cell line 2.3D which is made by expressing the c-myc oncogene in cloned embryonic day 10 primary neuroepithelial cells.
  • the neuroepithelial cell line is grown to approximately 50-90% and more preferably about 70% confluency and then the conditioned medium is collected at predetermined intervals. These intervals are those sufficient for HSPGs to be synthesized with a specificity for FGF-2 and then, following a change in GAG polymer composition and or structure, HSPGs are synthesized with specificity for FGF-1 at a later time.
  • FGF-2 specific HSPG is produced by 2.3D cells grown under non-confluent conditions whereas FGF-1 specific HSPG is produced by 2.3D cells grown under contact inhibiting conditions.
  • E9 or El 1 primary neuroepithelial brain cells are used, respectively.
  • the purification of the HSPG can be by any convenient means such as DEAE-Sepharose chromatography, affinity chromatography or immunosorbant chromatography amongst others.
  • Another aspect of the present invention contemplates an isolated core protein of a heparan sulfate proteoglycan (HSPG) wherein said core protein is capable of being substituted with GAG side chains such that one species of side chains is capable of preferentially binding to FGF-1 and another species is capable of preferentially binding to FGF-2.
  • HSPG heparan sulfate proteoglycan
  • isolated is meant a preparation of polypeptide or protein which has undergone at least one purification or separation step away from the naturally occurring environment of the polypeptide or protein.
  • isolated extends to a biologically pure preparation comprising at least 35%, preferably at least 45%, more preferably at least 55%, still more preferably at least 65%, even more preferably at least 75-80% and even more preferably greater than 95% of the polypeptide or protein as determined by weight, activity, immunoreactivity (e.g. antibody reactivity), cytokine binding activity or other convenient means.
  • the isolated polypeptide may be recombinant or synthetic or may be a non-full length molecule relative to the naturally occurring protein.
  • the polypeptide or protein of the present invention has, in a preferred embodiment, a molecular weight determined on SDS-PAGE of between 30 to 55 kDa. More specifically, the molecular weight is between 35 and 50 kDa and even more specifically is approximately 45 ⁇ 5 kDa.
  • the polypeptide or protein comprises a region having the amino acid sequence: G A S C E D C Q T F Y Y G D A Q R G T P Q D [SEQ ID NO:l] and/or a region having the amino acid sequence: G T P Q D C Q P C P C Y G A P R R T T P A [SEQ ID NO:2], or an amino acid sequence having at least 60%, more preferably at least 70%, even more preferably at least 80% and still more preferably at least 90% similarity to either or both of the above sequences or to a portion or region thereof.
  • the core protein bears some homology to the basement membrane protein proteoglycan, perlecan, although is of considerably smaller size (400 kDa versus 45 kDa) and has considerable higher glycosylation density. It also carries unique peptide domains and is encoded in an mRNA of approximately 3.5 kb.
  • the polypeptide or protein of the present invention is useful, for example, as a core substrate for GAG polymer synthesis to produce a specific cytokine binding molecule, such as an FGF-1 or FGF-2 binding molecule. Additionally, the polypeptide or protein may be used to generate antibodies against itself or related molecules or to generate agonists or antagonists to a naturally occurring form of the molecule. Most preferably, however, the polypeptide or protein will be in glycosylated form.
  • another aspect of the present invention relates to an isolated proteoglycan having one of at least two species of GAG polymer side chains such that one species binds preferentially to FGF-1 and another species binds preferentially to FGF-2.
  • proteoglycan is isolated from conditioned medium of a neuroepithelial cell line such as cell line 2.3D as hereinbefore described.
  • the neuroepithelial cell line may be transgenic for other genetic sequences and in particular those which modify or assist in the expression of the proteoglycan of the present invention.
  • the present invention as described herein is predicated in part on the discovery that different cytokines bind to the same proteoglycan depending on the composition and nature of the GAG side chains bound to the proteoglycan.
  • one form of an HSPG binds preferentially FGF-1 and another form of the same molecule binds preferentially to FGF-2.
  • the term "binds”, however, is not to be construed as imparting any limitation and extends to association, aggregation or any other form of interaction between molecules including tripartite interaction between an FGF, its receptor and the GAG side chain.
  • the proteoglycan or heparan sulfate polymer of the present invention will be useful in promoting, stimulating and/or enhancing activation of cytokines.
  • it is particularly exemplified by HSPG-FGF interaction required for presentation of FGF to the appropriate signal transducing receptors on neural precursor cells or any other cell type bearing the appropriate FGF receptor.
  • the GAG chains bearing specificity for FGF-2 also bear a carbohydrate subdomain which specifically bind a region of the FGFR1 receptor. In a particular embodiment, the GAG chains bind to the FGFRlIIIc receptor.
  • the heparan sulfate interaction with FGF-2 thus serves to activate the cytokine and directly couple it to its appropriate receptor by formation of a ternary complex.
  • the GAG chain with specificity for FGF-1 works in an analogous fashion with its particular FGF receptor.
  • a non-full length GAG polymer in promoting, stimulating and/or enhancing activation of cytokines such as FGF-1 or FGF-
  • the GAG polymers may be derivatised into smaller, functional fragments which are particularly efficacious in mediating FGF interaction. Accordingly, the present invention further contemplates mutants, derivatives, fragments, parts, homologues, analogues and chemical equivalents of the GAG polymers. Such forms are referred to collectively herein as "derivatives”.
  • Particularly preferred derivatives are fragments of GAG polymers obtainable by any number of means such as by chemical disruption and in particular acid hydrolysis with nitrous acid or by enzymatic cleavage with heparanase I and/or heparanase III
  • the derivatives contemplated herein may also act as antagonists and inhibit or reduce FGF-receptor interaction. Such antagonists may also have important therapeutic utility.
  • Non-full length GAG derivatives are particularly preferred as they are readily diffusable into tissues, have greater bioavailability, potentially exhibit greater specificity, tend to reduce adverse side effects and reduce the likelihood of adverse host immune reactivity.
  • the latter is particularly important since GAG polymers of non-human origin (e.g. from murine sources) are operative in humans and up to the present time, the preferred source of GAG polymers is from HSPGs isolated from murine sources.
  • the present invention contemplates GAG polymers or derivatives thereof from HSPGs of non-human origin (e.g. murine source) used in humans or non-murine animals (a heterologous system) as well as the use of a GAG polymer from an HSPG from the same species origin as the recipient of therapy (a homologous system).
  • An example of a derivative of a GAG polymer bearing both an FGF-2 binding domain and an FGF-2 receptor-binding domain is a 9 disaccharide unit fragment of a GAG polymer isolatable from an HSPG obtainable from 2.3D cells, grown under non- confluent conditions.
  • this 9 disaccharide units is further split into smaller fragments with heparanase I, the fragments are incapable of promoting cell-FGF-2 interaction, but are capable of blocking the formation of an activating FGF-2-heparan sulfate-FGF-2 receptor ternary complex.
  • the present invention also contemplates a similar fragment capable of interactivity with FGF-1.
  • "interactivity" includes functional interaction to facilitate FGF-receptor binding or alternatively antagonistic interaction to inhibit or reduce FGF-receptor interaction.
  • the present invention further contemplates a fragment of a GAG polymer, said fragment being at least about 5 disaccharides in length and obtained from a GAG polymer isolated from a HSPG from 2.3D cells grown under non-confluent conditions wherein said fragment is capable of interaction with FGF-2.
  • the fragment is isolated from a GAG polymer isolated from an HSPG from 2.3D cells grown under contact-inhibiting conditions and wherein said fragment is capable of interaction with FGF-1.
  • the fragment is at least about 7 disaccharides in length. More preferably, the fragment is at least about 9 disaccharides in length.
  • Another aspect of the present invention is directed to an antagonist of FGF-receptor interaction, said antagonist comprising a fragment of a GAG polymer, said fragment being at least about 3 disaccharides in length and obtained from a GAG polymer isolated from an HSPG.
  • the antagonist affects FGF-2-receptor interaction
  • it is from an HSPG from 2.3D cells grown under non-confluent conditions.
  • the antagonist affects FGF-1 -receptor interaction
  • it is from an HSPG from 2.3D cells grown under contact-inhibiting conditions.
  • the antagonistic fragment may also be at least about 5 or 7 disaccharides in length.
  • FGF-interactive derivatives may be readily detected by a number of convenient assays.
  • One such assay consists of a mitogenic assay on embryonic neuroepithelial cells, or the 2.3D cell line.
  • Another such assay is where labelled GAG fragments are chromatographed on FGF or FGF receptor (FGFR) peptide fragments coupled to an Affi-Gel 10 affinity support column and monitored for their ability to be retained.
  • Another procedure is a plate assay whereby appropriate amino acid fragments derived from either the FGF or the FGF receptor GAG-binding domains are derivatized to plastic and checked for their ability to bind appropriate [ 3 H]- or [ 35 S]- labelled GAG sequence.
  • Another aspect of the present invention contemplates a method of promoting, stimulating and/or enhancing interaction between a particular cytokine and a target site on a cell in an animal, said method comprising administering to said animal a GAG polymer or derivative thereof which preferentially binds to said cytokine, for a time and under conditions sufficient for said GAG polymer or its derivatives to promote binding of said cytokine with said target sequence.
  • the cytokine is FGF-1 or FGF-2 and the GAG polymer is GAGA and GAGB, respectively.
  • the effect of the GAG polymer is to maintain cells in a viable state.
  • the effect of the GAG polymer is to prevent or delay cell death.
  • two GAG polymers or derivatives thereof are administered simultaneously or sequentially to thereby promote interaction of at least two different cytokines with target sequences in the cell.
  • the present invention contemplates a method for inhibiting or reducing interaction between a particular cytokine and a target site on a cell in an animal, said method comprising administering to said animal an antagonist of cytokine- receptor interaction for a time and under conditions sufficient to inhibit or reduce said interaction.
  • the antagonist is a fragment of GAGB and inhibits or reduces FGF- 2-receptor interaction.
  • An example of such a fragment is a fragment of the 9 disaccharide unit fragment of GAGB.
  • a fragment of GAGA is used to inhibit FGF-1 -receptor interaction.
  • a method for promoting, stimulating and/or enhancing cell proliferation, migration and/or differentiation of any tissue which bears the appropriate FGF receptors or in an animal comprising the administration of a GAG polymer or derivatives thereof wherein said GAG polymer or its derivative interacts with FGF-1 or FGF-2 but not both.
  • This embodiment relates particularly to non-neuronal tissue.
  • the present invention provides a method for promoting or facilitating maintenance and survival of neuronal cells in an animal, said method comprising the administration of a GAG polymer or derivative thereof wherein said GAG polymer or its derivative interacts with FGF-1 or FGF-2 but not both. "Interacts" in this context is to facilitate FGF binding to its receptor.
  • the cells are motor neurons and the effect of the GAG polymer or its derivatives in combination with FGF 1 or 2 is to rescue motor neurons during the period of cell death.
  • the present invention extends to all neurons and in particular large neurons.
  • the GAG polymer or derivatives thereof are used in vitro to maintain or stimulate growth ⁇ f suitable cell lines, such as neuroepithelial cells.
  • the route of in vivo administration may be by any convenient means but is generally by intravenous administration.
  • Other forms of administration are possible, however, modification of the active molecules may be required to, for example, protect same from host enzymes or to facilitate passage through the blood vessel walls.
  • the effective amount of GAG polymer or derivative thereof will depend on the preparation, condition and host but, may generally be from at least about O.OOl ⁇ g/kg body weight to at least about lOmg/kg body weight. A more preferred range is at least about O.Ol ⁇ g/kg body weight to at least about lmg/kg body weight. Alternatively, a range of at least about l ⁇ g/kg body weight to about 500 ⁇ g/kg body weight. Administration may be a single dose or a series of doses. Additionally, the GAG polymer or derivatives thereof may also be complexed with an FGF.
  • compositions comprising a GAG polymer or derivative thereof capable of interactivity with FGF-2, said composition further comprising one or more pharmaceutically acceptable carriers and/or diluents.
  • the pharmaceutical composition comprises a GAG polymer or derivative thereof capable of binding FGF-1.
  • the composition comprises at least two species of GAG polymers or derivatives thereof or one species of GAG polymer and a cytokine or other active molecule.
  • a heparan sulfate polymer linked to a particular core molecule capable of targeting the polymer to a specific site or group of sites.
  • a hybrid molecule will be particularly useful, for example, for localized FGF treatment.
  • the present invention extends to heterologous and homologous systems in relation to the species from which the HSPG is purified and the intended recipient, for example, murine heparan sulfate polymer is active both in human cells and in chick embryos amongst other animal tissues.
  • murine or human HSPGs are used as a source of GAG polymers.
  • Still another aspect of the present invention contemplates a method for rescuing neurons during the period of cell death in a mammal, said method comprising administering to said mammal an effective amount of a GAG polymer or a derivative thereof from an HSPG obtainable from embryonic day 8-10 cells and wherein said GAG polymers or derivative is capable of interaction with FGF-2 but not FGF-1.
  • the neurons are large neurons. More particularly, the neurons are motor neurons.
  • a further aspect of the present invention provides a method for promoting the viability of cells carrying an FGFRlIIIc receptor for FGF-2 in a mammal, said method comprising administering to said mammal an effective amount of a GAG polymer or a derivative thereof from an HSPG obtainable from embryonic day 8-10 cells and wherein said GAG polymers or derivative is capable of interaction with FGF-2 but not FGF-1.
  • the mammal is a human and preferably the cells are 2.3D cells grown under non-confluent conditions.
  • the present invention extends to the use of the GAG polymers of the present invention in situations where cells producing FGF-1 and or FGF-2 are transplanted into brain parenchyma to relieve the symptoms of neurological disorders such as Huntington' s Disease or Parkinson's Disease or afflictions which involve Parkinsonism.
  • the cells are embryonic cells and the GAG polymers facilitate FGF interaction with its receptors.
  • the embryomc cells are genetically engineered to express an FGF such as FGF-1 and or FGF-2.
  • E14 embryonic cells may be used.
  • the cells are then transplanted into brain tissue to an area generally occupied by cells of the substantial nigra which are dopaminergic. After, during or prior to grafting, GAGA and/or GAGB is supplied which will greatly increase the benefits of the transplanted cells both anatomically and behaviourally.
  • another aspect of the prevent invention contemplates a method of ameliorating the effects or symptoms of neurological disorders in a mammal, said method comprising transplanting cells which synthesize FGF-2 to a neurological environment and contacting said transplanted cells with an effective amount of a GAG polymer capable of interaction with FGF-2 but not FGF-1 and obtainable from an HSPG from murine cells at approximately embryomc day 8-10.
  • a GAG polymer capable of interaction with FGF-2 but not FGF-1 and obtainable from an HSPG from murine cells at approximately embryomc day 8-10.
  • the GAG polymer is GAGB.
  • a further aspect of the present invention contemplates a method of ameliorating the effects or symptoms of neurological disorders in a mammal, said method comprising transplanting cells which synthesize FGF-1 to a neurological environment and contacting said transplanted cells with an effective amount of a GAG polymer capable of interaction with FGF-1 but not FGF-2 and obtainable from an HSPG from murine cells at approximately embryomc day 11-13.
  • the GAG polymer is GAGA.
  • the cells are first genetically engineered to express increased amounts of an FGF or a derivative thereof.
  • Yet another aspect of the present invention contemplates a method of ameliorating the effects or symptoms of neurological disorders in a mammal, said method comprising transplanting cells genetically engineered to express FGF-2 to a neurological environment and contacting said transplanted cells with an effective amount of a GAG polymer capable of interaction with FGF-2 but not FGF-1 and obtainable from an HSPG from murine cells at approximately embryonic day 8-10.
  • the GAG polymer is GAGB.
  • the present invention provides a method of ameliorating the effects or symptoms of neurological disorders in a mammal, said method comprising transplanting cells genetically engineered to express FGF-1 to a neurological environment and contacting said transplanted cells with an effective amount of a GAG polymer capable of interaction with FGF-1 but not FGF-2 and obtainable from an HSPG from murine cells at approximately embryonic day 1 1-13.
  • a GAG polymer capable of interaction with FGF-1 but not FGF-2 and obtainable from an HSPG from murine cells at approximately embryonic day 1 1-13.
  • the GAG polymer is GAGA.
  • preferred animals for treatment are mammals such as humans, livestock animals (e.g. sheep, cows, pigs, horses), companion animals (e.g. dogs, cats) or laboratory test animals (e.g. mice, rats, rabbits). Most preferably, the mammal is a human.
  • Figure 1 is a graphical representation of the results of affinity chromatography using HSPG coupled to an Affi-Gel 10 (see Example 1). Values presented are the means and SDs of six determinations from two to four experiments.
  • Figure 2 is a graphical representation showing gel filtration analysis of GAG chains from E9 and El l HSPGs on BioGel P-2 columns.
  • CS chondroitin sulfate
  • Figure 3 shows [ 3 H]thymidine incorporation into neuroepithelial cells maintained in either FGF1 or FGF2 with supplemental glycosaminoglycan.
  • E10 neuroepithelial cells were trypsinized (0.1% w/v trypsin) to remove surface and adherent proteoglycans, allowed 2 hours to recover, and then plated onto HL-a plates in the presence of 5 ng/ml of FGF-1 or FGF-2 at a cell density of 1,500 cells per well (1). After 36 hours the cells were pulsed for 16 hours with [ 3 H]thymidine, harvested, washed, and counted.
  • E9 and El 1 HSPG preparations were mixed with anti-2.3D core protein antibody (10:1 v/v for two hours), mixed with Pansorbin (CalBiochem, 10:1 v/v for two hours), clarified, and the procedure repeated.
  • Experiments with ⁇ SO ⁇ labelled HSPGs demonstrated that greater than 88% of HSPGs are removed by this procedure.
  • Equivalent volumes of the immunodepleted HSPGs were then added back to the cultures. Values are the means and standard deviations (SDs) of six determinations.
  • Figure 4 is a graphical representation of the dose-response relationship between increasing amounts of glycosaminoglycan fragments (X axis) and their effects on 2.3D cell proliferation (Y-axis) as monitored by [ 3 H]-thymidine uptake (measured in cpm) after 24 hour exposure in tissue culture to a fixed concentration of FGF-2(5ng ml. in the dish), A DDS, no activity; ⁇ — ⁇ ODS, activity; • ⁇ " • - • • • HSPG , heparan sulphate.
  • Figure 5 is a graphical reproduction demonstrating that both the 6 disaccharide unit (12 mer, “DDS”) and the 3 disaccharide unit (6 mer, “HeS”) are capable of competitively inhibiting the mitogenic effects (as monitored by thymidine uptake in cpm) of the 9 disaccharide unit (18 mer, "DDS") on 2.3D cells in culture grown in a fixed concentration of FGF-2(5ng/ml).
  • Figure 6 is a graphical representation demonstrating further the specificity of the interaction between the 9 disaccharide unit (ODS; 18 mer) and the FGF receptor type 1 isoform.
  • 2.3D cells were grown as before in culture in the presence of FGFs, sugars and receptor-blocking peptides. The curve marked FGF-2/ODS establishes the baseline positive control.
  • Figure 7 is a graphical representation showing rotational behaviour of grafted rats following amphetamine administration. Only rats implanted with FGF-2 in combination with GAGB showed a significant drop in the turning response induced by amphetamine.
  • FGF-2 specific HSPG is derived from the conditioned medium produced by the neuroepithelial cell line 2.3D, previously made by expressing the c-myc oncogene in embryonic day 10 primary neuroepithelial cells.
  • the 2.3D neuroepithelial cell line is grown to 70% confluency, the cells pulsed with overnight, the medium conditioned collected and clarified by centrifugation, and then passed through DEAE- Sepharose equilibrated in Tris-buffered saline (pH7.4).
  • the column is washed with 10 column volumes of 0.25 M NaCi 0.1% v/v Triton X-100, the same wash supplemented with 8 M Urea, then 0.3 M sodium formate (pH 3.5)/8 M Urea, and then with 0.05 M Tris-HCl (pH 8)70.01% v/v Triton X-100.
  • HSPGs are released from the column with an increasing gradient of NaCl (0.15-1.0 M) in 0.01 M Tris-HCl (pH 8)/0.01% v/v Triton X-100.
  • a similar purification protocol is employed in the preparation of FGF-1 - specific HSPG except that the 2.3D cells are cultured under contact-inhibiting conditions by growing the cells to 100% confluency and then maintaining the cells under these conditions for 6 days before collecting the conditioned medium.
  • a suitable method for growing cells to confluency is described in reference 13.
  • the proteoglycans were sized after Sepharose CL-6B gel chromatography.
  • the proteoglycans present eluted as a single peak at approximately MW 450,000.
  • the proteoglycan peak disappeared after both nitrous acid (pH 1.5) and heparanase III treatment, but not chondroitinase ABC.
  • the proteoglycan is thus a heparan sulfate.
  • the core protein of the proteoglycan ran on SDS-PAGE gels at approximately 45,000.
  • the side chains derived from non-confluent 2.3D cells averaged 20,000 daltons.
  • the core protein from this proteoglycan was treated with trypsin, carboxymethylated in 6M guanidine HC1 (pH 8.6), reduced with beta-mercaptoethanol (50°C, N2, 1 hour), alkylated, dialyzed against 5% v/v acetic acid, chromatographed on Biogel P10 and run on a reverse phase HPLC Zorbax OD5 and the fragments sequenced for amino acids on a gas phase sequenator.
  • the core protein was also subjected to V8 protease for 6 hours, run on Sepharose 4B (0.2 M NaC170.02 M Tris-HCl (pH 8)), then on DEAE-5PW HPLC columns, reduced with dithiothreitol, alkylated with iodoacetamide, rerun on DEAE-5PW HPLC and the peaks similarly amino acid sequenced. Partial amino acid sequence obtained from the procedure is shown in Table 1.
  • HSPG For preparation of HSPG in a particular glycosylated form for binding to either FGF-1 or FGF-2, purification can conveniently occur for Ell and E9 neuroepithelial cultures (see Example 2), respectively.
  • Serum-free media conditioned over 2.3D cells, E9, or El 1 (see below) neuroepithelial cell cultures (10 ⁇ cells per 16-mm well for 24 hours) were filtered through 0.45 ⁇ mesh and chromatographed through a low-pressure Econo-Pac Q Sepharose cartridge (Bio- Rad) at 2 ml/min. The column was washed with 0.15 M TBS, pH 7.4, until the absorbance at 280 nm reached baseline. The bound material was then released with an NaCl gradient from 0.15 to 1.0 M and collected in 3-ml fractions.
  • the cells were maintained in DMEM containing in order to detect GAG side chains; in other experiments the cells were maintained in DMEM containing [ 3 ⁇ S]methionine in order to detect HSPG core proteins.
  • Purified HSPG preparations from either E9 or Ell conditioned media, or 2.3D cell media were ligated to the affinity agarose support Affi-Gel 10 (Bio-Rad) in carbodiimide coupling buffer according to the manufacturers instructions. Approximately 100 ⁇ g of each HSPG preparation was bound to each 1 ml column volume of gel bed. The bound support was then decanted into small chromatographic columns and washed in 0.15 M Tris-buffered saline (TBS, pH 7.4).
  • the inventors discovered that neuroepithelial cells differentially regulate the expression of FGF during development. Studies were performed on mesencephalic and telencephalic neuroepithelial tissue at embryonic day 9, 10, 11 and 13 and this tissue is referred to herein as E9, E10, El l and El 3, respectively. In particular, the inventors showed FGF-2 expression in E9 and then subsequently FGF-1 expression in El l.
  • conditioned media were collected from E9 and El l cells maintained in either [ ⁇ S]methionine to label the core protein or in -"SO4 to label the GAG side chains.
  • E9 and El 1 HSPGs were stripped of GAGs with heparitinase, the core proteins appeared to have very similar molecular weights of about 45 kDa (Fig. 2A). This similarity was further substantiated by immunoprecipitation with an affinity-purified rabbit antibody raised against the core protein of the nonconfluent 2.3D HSPG.
  • This antibody was able to precipitate core proteins with an identical molecular weight to those obtained from the original DEAE isolates from both E9 and El l (Fig. 2A) and 2.3D cells. These molecular weights together with preliminary amino acid sequencing of core protein fragments indicate that neuroepithelial cells secrete a single unique species of HSPG. When these core proteins were digested with trypsin, the resultant peptides yielded profiles on SDS-polyacrylamide gels and reversed phase high pressure liquid chromatography that were essentially identical.
  • HSPG-binding specificity coincides with the ability of each factor to stimulate cell proliferation
  • El l neuroepithelial cells were isolated, pretreated with trypsin to exogenous HSPGs and attached growth factors, and then exposed to either FGF-1 or FGF-2 in the presence of HSPGs obtained from E9 or El l neuropithelium (Fig. 3).
  • E l l HSPG was approximately four times more effective with FGF-2. This response to the HSPGs was dose-dependent within the range 0.01 to lO ⁇ g/ml.
  • Heparan sulfate polymers in the form of GAG chains of the E9 and El 1 HSPGs were prepared from a 100 ⁇ l sample of immunopurified proteoglycan layered onto a BioGel P-2 column equilibrated in TBS, collected in the void volume, and digested with 1 mg ml Pronase for 4 hours at 25°C. The samples were concentrated to 50 ⁇ l by dialysis against solid polyethylene glycol at 25°C for 2 hours, adjusted to 4 M guanidinine hydrochloride/50 mM Tris, pH 7.0. The eluted fractions were counted in Aquasol (NEN, Dupont, Sydney).
  • the column was calibrated with samples of ⁇ C-labelled dextran (70 kD), [ 3 H] chondroitin sulphate (50 kD) and [ 3 H]heparin (12 kD).
  • the purification of the FGF-1- and FGF-2- specific heparan sulfate polymers is shown in Figure 2.
  • the GAG side chains were in some cases repurified through Q Sepharose using similar procedures to those for total HSPG following the methods of Cole and Burg (8) and Kojima et al (9).
  • the development of the vertebrate nervous system is characterized by an initial overproduction of neurons in many regions followed by their large-scale elimination. This phenomenon takes place at a particularly important stage during the development of embryonic neurons, the period immediately following the arrival of their axons in the specific target fields.
  • Ideas current in neurotrophic theory place the basis of this cell death on a competition for limiting amounts of crucial trophic factors supplied by the target organ.
  • only two defined trophic molecules have been shown to support embryonic neuronal survival in vivo - nerve growth factor and brain-derived neurotrophic factor.
  • somatic motor neurons of the spinal cord undergo naturally occurring cell death during embryonic development.
  • motor neurons are insensitive to NGF there is circumstantial evidence that the survival of embryomc motor neurons is dependent on trophic substances within developing skeletal muscles.
  • Skeletal muscle contains substances that enhance the survival and development of motor neurons in vitro.
  • the inventors used the optical dissector of Gundersen et al. (11) to estimate the total number of neurons in the developing chick lumbar lateral motor column and to examine the effects of growth factors (FGF-2, CNTF, LIF, NGF) on neuronal number.
  • FGF-2, CNTF, LIF, NGF growth factors
  • the effect of FGF-2 alone or complexed with heparan sulfate polymers from E9 HSPG was investigated in the chick embryo spinal cord model.
  • White Leghorn chick embryos were treated daily in ovo with either 0.9% w/v saline or purified growth factor in saline from E6 to E9. Each growth factor in a volume of 50 ⁇ l. was applied to the vascularized chorioallantoic membrane through a window in the shell as described by Oppenheim et al. (12).
  • Embryos treated with FGF-2 received either daily applications of 2 ug of recombinant human FGF-2 in 50 ul of 0.9% w/v saline, or the same FGF-2 that had been mixed on an orbital shaker with purified E9 HSPG-GAG chains at a molar ratio of 3:1 (GAG:FGF-2) in eppendorf tubes at room temperature for 2 hours prior to application to the vascularized chorioallantoic membrane.
  • the spinal cords were immersion fixed in Carnoy's fixative for 1 h, dehydrated in 100% w/v ethanol overnight and embedded in glycolmethacrylate (Polaron Embedding Medium, Bio Rad.).
  • FGF-2-specific GAG polymer was subjected to compositional analysis as follows:
  • Neuroepithelial cells were grown in 0.5 ml 10% v/v FCS/DMEM and 2 ng/ml FGF-2 in 24 well tissue culture plates at a density of 100,000 cells/well. The cells were allowed to settle in a 10% v/v CO/air-humidified incubator for 30-60 min before addition of 20 uCi/ml [H 3 ]glucosamine. Wells were monitored daily for contamination or excessive cell death (over 50%) and those cells and media discarded. Cultures were further incubated for 50-60 hours. The medium was gently removed and centrifuged (1000 rpm for 5 min) to remove any cell debris and stored at -20°C until required.
  • the conditioned media was then subjected to ion-exchange chromatography on a DEAE- Sephacel column (2 ml) which had previously been blocked with heparin and equilibrated in 150 mM NaCl with phosphate buffered saline, pH 7.2.
  • the sample was then washed with ten column volumes of 250 mM NaCl in 50 mM phosphate buffered saline, pH 7.2.
  • the bound material (primarily heparan sulfate, chondroitin sulfate and dermatan sulfate) was eluted in a step elution at 1 M NaCl in 50 mM phosphate buffered saline, pH 7.2 and 2 ml fractions collected.
  • Fractions containing the tritiated glucosamine (primarily fractions 1-3) were pooled and desalted on Amicon concentration cones, freeze dried and resuspended in miiiimal volume (100-500 ul maximum). Sialic acid was removed with neuraminidase in 25 mM Na-acetate pH 5.0, for 4 hours. Chondroitin sulfate and dermatan sulfate were digested with chondroitin ABC lyase treatment for 4 hours at 37°C and a further digest overnight with fresh enzyme.
  • the core protein and all of the lyases are digested with Pronase at 37°C for 24 h and the sample passed through a 2 ml dEAE-Sephacel column and eluted as previously described while collecting 1 ml fractions. The sample was finally desalted on a 1 cm x 35 cm P2 column and the Vo fraction collected and freeze dried for further analysis.
  • Heparitinase (heparitinase I), heparitinase II and heparitinase IV were used at a concentration of 25 m units ml in 100 mM-sodium acetate/0.2 mM-calcium acetate, pH 7.0. Samples were incubated at 37°C for 16 h and then a second aliquot added and incubated a further 4 hours. Heparinase was used at a concentration of 50 m units/ml in the same buffer as heparitinase.
  • Disaccharide composition was analysed by either complete depolymerisation of the entire heparan sulfate chain with heparitinase, heparitinase II, heparitinase IV and heparinase (yields >/+ 95%) or subjected to nitrous acid (pH 1.5) so that both the disaccharide fraction and the tetrasaccharide fraction could be separately collected. These pools were freeze dried and resuspended in 300 ul water. The disaccharides or tetrasaccharides were then separated by SAX-HPLC on either one or two ProPac PA1 analytical columns (4 x 250 mm; Dionex, Surrey, United Kingdom).
  • Radiolabelled heparan sulfate that had been treated with a variety of reagents was mapped by gradient PAGE as described previously by Turnbull and Gallagher (1988) with some modifications. Briefly, 25-33% w/v -polyacrylamide-gradient gels (32cm x 16cm x 0.75mm) were prepared with a 5% w/v stacking gel. Samples were electrophoresed as previously described until the phenol red marker was about 1 cm from the bottom. Gel was equilibrated in 10 mM Tris/acetate buffer containing 0.5 mM- EDTA for 10-20 min.
  • Oligosaccharides were then transferred onto a positively charged nylon membrane (Biotrace RP) in a Trans-blot tank at low voltage in the same buffer for 3-4 hours.
  • the oligosaccharides were detected by fluorography of the membrane by using Enhance surface autoradiography enhancer and Kodak X-Omat AR X-ray film.
  • the compositional analysis of the FGF-2-specific GAG polymer is shown in Table 3.
  • Figure 4 is a graphical representation of the dose-response relationship between increasing amounts of glycosaminoglycan fragments (X axis) and their effects on 2.3D cell proliferation (Y-axis) as monitored by [ 3 H]-thymidine uptake (measured in cpm) after 24 hour exposure in tissue culture to a fixed concentration of FGF-2(5ng/ml, in the dish).
  • X axis glycosaminoglycan fragments
  • Y-axis cell proliferation
  • [ 3 H]-thymidine uptake measured in cpm
  • ODS octadecasaccharide
  • Figure 5 demonstrates that both the 6 disaccharide unit (12 mer, "DDS”) and the 3 disaccharide unit (6 mer, "HeS”) are capable of competitively inhibiting the mitogenic effects (as monitored by thymidine uptake in cpm) of the 9 disaccharide unit (18 mer, "ODS”) on 2.3D cells in culture grown on a fixed concentration of FGF-2(5ng/ml).
  • DDS disaccharide unit
  • HeS 3 disaccharide unit
  • Figure 6 demonstrates further the specificity of the interaction between the 9 disaccharide unit and the FGF receptor type 1 isoform.
  • 2.3D cells were grown as before in culture in the presence of FGFs, sugars and receptor-blocking peptides.
  • the curve marked FGF-2/ODS establishes the baseline positive control.
  • the subsequent experiments are performed in the presence of increasing concentrations of the peptide "K22", a 22 amino acid peptide established by Kan et al. (15) to represent the portion of the FGFR1, designated Ig domain 2, which engages the sugar before the FGF docks with the receptor in the Ig3 domain.
  • the second curve shows that cells grown in the presence of the E12 (ie.
  • FGF-1 specific HSPG and FGF-1 cannot be inhibited in their growth by the presence of the FGFR1 -specific K22K peptide. Therefore the FGF-1- specific GAG is not using this receptor for signal transduction, unlike FGF-2.
  • the next curve, designated ODS/FGF-1/FGFR1 also shows a lack of inhibition of growth, demonstrating both that the ODS does not potentiate FGF-1 (the growth plateau is lower than for the first 2 curves), and that K22K FGFR1 -specific peptides have no effect on this growth.
  • the last curve, marked ODS/FGF-2/FGFR1 shows increasing inhibition of growth in the presence of increasing amounts of the FGFR1 -specific peptide.
  • the ODS stimulates the effects of FGF-2 specifically, and does so through the FGFR1 receptor.
  • Pathogen-free Sprague Dawley rats (body weight 158-163 g) were kept under regular day and night conditions at constant 23 °C temperature with free access to food pellets and water.
  • the toxin was dissolved at a concentration of 6 mg/ml in saline and 1.5 ul was injected at 4.3 mm posterior to bregma, 1.5 mm laterial and 7.3 mm below dura, the syringe was raised 0.2 mm and another 1.5 ul was injected at -7.1 mm.
  • a canula connected to an osmotic Alzet minipump (model 2002; Alza Corporation, Palo Alto CA) was then implanted in the brainstem immediately following the lesion; the pumps were loaded with one of four test substances listed below. Approximately one week after the lesion, control uninfused but chemically lesioned rats were tested for turning behaviour with amphetamine sulfate (5 mg/kg).
  • Infused rats were divided into groups that were matched for rotational scores: infusion with saline (phosphate-buffered saline: PBS) vehicle alone; infusion with saline plus brain-derived neurotrophic factor (BDNF; 100 ng/ml); infusion with saline plus FGF-2 (100 ng/ml); or infusion with FGF-2 supplemented with GAGB (10 ug ml). Results are shown in Figure 7.
  • mice were deply anaesthetised and perfused with 4% v/v paraformaldehyde in 100 mM PBS. Brains were removed, sectioned coronally at 40 um on a freezing microtome, stained for tyrosine hydroxylase or Nissl substance and quantitated sterologically according to the methods of Janson and Moller (18).
  • GAGB may be used in any situation where cells synthesize FGFs including cells genetically engineered to express FGFs, are transplanted into brain parenchyma to relieve the symptoms of neurological disorders such as Huntington' s Disease or Parkinson's Disease, or afflictions which involve parkinsonism. In this disease state, where there is loss of dopaminergic cells of the substantial nigra, transplanted cells have been shown to ameliorate the behavioural motor deficits which ensue (17).
  • One potential method for increasing the viability of dopamine neurons after grafting may be to supply the cells with trophic support such as that supplied by FGF-2.
  • the FGF-2 cDNA spliced into a retroviral vector under the control of a promoter such as the long terminal repeat (LTR) or a constitutive promoter such as actin, accompanied by an antibiotic resistant gene, may be used to stably transfect suitable cells.
  • a promoter such as the long terminal repeat (LTR) or a constitutive promoter such as actin, accompanied by an antibiotic resistant gene
  • the FGF cDNA may contain additional sequences from pre-pro regions of secreted growth molecules such as nerve growth factor to enhance the extracellular secretion of the FGF.
  • the present invention demonstrates that supplying the grafted cells with additional glycosyaminoglycans that selectively couple FGF-2 with FGFR1 greatly increases the viability of such grafts. Furthermore, supplying GAGB to any such neural transplant expressing FGF will greatly increase the benefits of such grafts, both anatomically and behaviourally.
  • the grafts may involve non-neural cells such as fibroblasts to carry the FGF-2 gene into the damaged brain tissue. This will be a useful strategy for enhancing the clinical effectiveness of dopaminergic treatments based on FGF-2 neurotrophic activity.
  • Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications.
  • the invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Abstract

La présente invention concerne d'une manière générale une molécule thérapeutique. Plus particulièrement, la présente invention concerne une molécule thérapeutique constituée d'un polymère héparane-sulfate ou d'un dérivé de celui-ci, que l'on peut obtenir à partir de l'héparane-sulfate protéoglycane et qui est capable d'avoir une interaction avec une cytokine spécifique. La molécule thérapeutique de la présente invention est utile pour favoriser la fonction de la cytokine in vitro et in vivo. La présente invention concerne plus particulièrement les effets de polymères d'héparane-sulfate sur les facteurs de croissance des fibroblastes.
PCT/AU1996/000034 1995-01-27 1996-01-25 Molecule therapeutique WO1996023003A1 (fr)

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WO2014193308A1 (fr) 2013-05-27 2014-12-04 Agency For Science, Technology And Research Sulfate d'héparane
WO2015167401A1 (fr) 2014-04-30 2015-11-05 Agency For Science, Technology And Research Héparane sulfates
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

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