WO2009005353A1 - Composés d'oligosaccharide de liaison élevée, compositions et leurs utilisations - Google Patents

Composés d'oligosaccharide de liaison élevée, compositions et leurs utilisations Download PDF

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WO2009005353A1
WO2009005353A1 PCT/NL2008/050443 NL2008050443W WO2009005353A1 WO 2009005353 A1 WO2009005353 A1 WO 2009005353A1 NL 2008050443 W NL2008050443 W NL 2008050443W WO 2009005353 A1 WO2009005353 A1 WO 2009005353A1
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compound according
toxin
och
gml
dendrimer
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Hubert Philippe Endzt
Alexander Franciscus Van Belkum
Bartholomeus Casper Jacobs
Teris Van Beek
Gerben Visser
Johannes Teunis Zuilhof
Roelof Jan Pieters
Huibert Marinus Branderhorst
Robertus Matthias Joseph Liskamp
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Erasmus University Medical Center Rotterdam
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002—Dendritic macromolecules
    • C08G83/003—Dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/641—Branched, dendritic or hypercomb peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/28—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Vibrionaceae (F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates

Definitions

  • the invention relates to oligosaccharide compositions having affinity for proteins and the medical or biotechnological or diagnostic use of such compositions.
  • Gangliosides are a class of glycolipids, often found in cell membranes, which consists of three elements. One or more sialic acid residues are attached to an oligosaccharide or carbohydrate core moiety, which in turn is attached to a hydrophobic lipid (ceramide) structure which generally is embedded in the cell membrane.
  • the ceramide moiety includes a long chain base (LCB) portion and a fatty acid (FA) portion.
  • LCB long chain base
  • FA fatty acid
  • Gangliosides are classified according to the number of monosaccharides in the carbohydrate moiety, as well as the number and location of sialic acid groups present in the carbohydrate moiety. Monosialogangliosides are given the designation "GM”, disialogangliosides are designated “GD”, trisialogangliosides “GT”, and tetrasialogangliosides are designated "GQ”. Gangliosides can be classified further depending on the position or positions of the sialic aeid residue or residues bound.
  • Gangliosides are most abundant in the brain, particularly in nerve endings. They are believed to be present at receptor sites for neurotransmitters, including acetylcholine, and can also act as specific receptors for other biological macromolecules, including interferon, hormones; viruses, bacterial toxins, and the like. Gangliosides have been used for treatment of nervous system disorders, including cerebral ischemic strokes. See, e.g., Mahadnik et al. (1988) Drug Development Res. 15: 337-360; U.S. Pat. Nos. 4,710,490 and 4,347,244; Horowitz (1988) Adv. Exp. Med. and Biol. 174: 593-600; Karpiatz et al.
  • gangliosides are found on the surface of human hematopoietic cells (HMebrand et al. (1972) Biochim. Biophys. Acta 260: 272-278; Macher et al. (1981) J. Biol. Chem. 256: 1968-1974; Dacremont et aL Biochim, Biophys. Acta 424: 315-322; Klock et al. (1981) Blood CeIk 7: 247) which may play a role in the terminal granulocytic differentiation of these cells. Nojiri et al. (1988) J. Biol. Chem. 263: 7443-7446. These gangliosides, referred to as the "neolacto" series, have neutral core oligosaccharide structures having the formula [GaLbeta.-
  • Ganglioside "mimics” are often associated with some pathogenic organisms.
  • the core oligosaccharides of low-molecular-weight Iipo Po ⁇ y Saccharide (LPS) of Campylobacter jejuni 0:19 strains were shown to exhibit molecular mimicry of gangliosides. Since the late 1970s, Campylobacter jejuni has been recognized as an important cause of acute gastroenteritis in humans (SMrrow (1977) Brit. Med. J. 2: 9-11).
  • Epidemiological studies have shown that Campylobacter infections are more common in developed countries than Salmonella infections and they are also an important cause of diarrheal diseases in developing countries (Nachamkin et al.
  • LPS lipopolysaccharide
  • Terminal oligosaccharide moieties identical to those of GDIa, GD3, GMl and GTIa gangliosides have been found in various C. jejuni 0:19 strains.
  • C. jejuni OH4384 belongs to serotype 0:19 and was isolated from a patient who developed the Guillain-Barre syndrome following a bout of diarrhea (Aspinall et al. (1994a), supra.). It was shown to possess an outer core LPS that mimics the tri-sialylated ganglioside GTIa. Molecular mimicry of host structures by the saccharide portion of LPS is considered to be a virulence factor of various mucosal pathogens which would use this strategy to evade the immune response (Moran et al. (1996a) FEMS
  • Towson et al support the view that immunoadsorption plasmapheresis could potentially be developed for the acute depletion of serum anti-GMl antibodies in patients with neuropathic disease, and also provide purified human anti-GMl antibodies for analytical studies.
  • the cholera toxin (CT) is a prime example of a multivalent protein and is capable of binding simultaneously to the carbohydrate moieties of five GMl gangliosides.
  • the binding of the five B subunits is of critical importance for the internaHzation and subsequent disease process of this AB ⁇ toxin (E. Pan, et al., Curr, Opin, Struct. Biol. 2000, 10, 680-686).
  • the development of strong binding agents to the toxin is of interest for the development of disease prevention/treatment, but also for the detection of toxin in patient samples or in materials suspect of terrorist origin (S, Ahn-Yoon, et al., Anal. Chem.
  • the invention provides high binding oligosaccharide compounds and compositions comprising one or more of said compounds.
  • the high binding oligosaccharide compounds provided herein are preferably synthetic ganglioside mimics designed to detect, purify, characterize and/or deplete substances, e.g. bacterial toxins and serum antibodies, that are related to human diseases. They consist of an oligosaccharide structure that is essentially identical to the carbohydrate portion of a ganglioside, attached to aU ⁇ yi spacer molecules, which in turn are covalently linked to multivalent, such as divalent, tetravalent or octavalent dendrimers.
  • Dendrimers are molecules that at least partially consist of repeating units, and which are molecules that have a two- or three- dimensional diverting branching structure starting from the "base" of the dendrimer. The name is derived from the Greek word for tree; dendrimers are (often) tree-shaped molecules.
  • the compound preferably has a terminal azide, or other type of reactive group, by which it can be covalently bound to a solid carrier or solid surface, such as a bead, ELISA plate, monolithic column, monolitenic particles or other solid carrier that can be used to detect or adsorb ligands for ganglioside-ligands, including bacterial toxins and serum anti-ganglioside antibodies.
  • a solid carrier or solid surface such as a bead, ELISA plate, monolithic column, monolitenic particles or other solid carrier that can be used to detect or adsorb ligands for ganglioside-ligands, including bacterial toxins and serum anti-ganglioside antibodies.
  • a solid carrier or solid surface such as a bead, ELISA plate, monolithic column, monolitenic particles or other solid carrier that can be used to detect or adsorb ligands for ganglioside-ligands, including bacterial toxins and serum anti-ganglioside antibodies
  • said carrier is selected from the group of beads, nano-beads or nanoparticles, monoliths, Elisa plates, Si wafers, capillaries and glass slides, and other carriers known in the art.
  • said carrier comprises a methacrylate-based monolith, such as one comprising polymethacrylate-based polymer.
  • the invention also provides a highly effective and selective immunoadsorption because of the use of the preferred authentic ganglioside oligosaccharide sequences that are the most physiological and relevant targets in diseases. Further the use of an extremely high density of these oligosaccharide sequences required for multivalent binding and for the conformational epitopes mimicking those in the physiological clusters of gangliosides in lipid rafts at the neural membranes is provided herein, as well as the use of multivalent dendrimeric scaffolds that further increase the epitope density, or the use of elongated spacer arms and in a preferred embodiment the use of the monolith composites resulting in an strong increase of efficient surface area for antibody-ligand interaction.
  • the invention provides a compound or substance having multivalent binding capacity for a protein, said compound comprising at least two oligosaccharide moieties linked to a dendrimer, preferably having at least three, or more preferably, four oligosaccharide moieties linked to a dendrimer. It is most preferred for authenticity that at least one of said oligosaccharide moieties is a monosialogangliosid (GM), a disialogangliosid (GD), a trisialogangliosid (GT), a tetrasialogangliosid (GQ) or a mimic thereof. It is preferred that at least one of said oligosaccharide moieties has been linked to said dendrimer via 'click' chemistry (Also described in EP 1 733 742).
  • such a compound according to the invention is linked to a dendrimer carrier.
  • Preferred dendrimers are selected from the group of hydrophilic dendrimers, particularly wherein said dendrimer comprises oxy-ethylene spacer arms. It is preferred that at least one of said oligosaccharide moieties linked via a spacer arm to a dendrimer, preferably selected from the group of spacers consisting of 11 to 80 atoms.
  • said spacer arm comprises C, H, O, and N atoms and preferably consists of 34 atoms.
  • a further aspect of the invention relates to a method for providing a multivalent compound of the invention, and to a method for immobilizing said compound to a solid carrier. Also provided are compounds and solid carriers obtainable by such methods.
  • the invention herewith provides a compound or composition having multivalent binding capacity for a bacterial toxin, preferably an ABs toxin such as Cholera toxin (CT), E. coli heat labile toxin (LT) or Shiga toxin (Stx), and use of such a compound or composition for the detection of a bacterial toxin, preferably an ABs toxin in a sample.
  • CT Cholera toxin
  • LT E. coli heat labile toxin
  • Stx Shiga toxin
  • the invention also provides use of such a compound for the preparation of a pharmaceutical composition, preferably for treatment of a subject suspected to be suffering of an intoxication with a bacterial toxin such as an ABs toxin. Preventive treatment of subjects prone to such intoxication is herein also provided.
  • the invention also provides use of such a composition for the preparation of a pharmaceutical composition for treatment of a subject suspected to be suffering from an autoimmune disease, for example for treatment of a subject suspected to be suffering from an autoimmune neuropathy, such as wherein said neuropathy comprises Guillain Barr ⁇ Syndrome.
  • Preventive treatment of subjects prone to such autoimmune disease is herein also provided.
  • the invention thus herewith also provides a pharmaceutical composition comprising one or more compounds according to the invention having multivalent binding capacity for a bacterial toxin, preferably an ABe toxin such as Cholera toxin (CT), E. coli heat labile toxine (LT) or Shiga toxin (Stx).
  • CT Cholera toxin
  • LT E. coli heat labile toxine
  • Stx Shiga toxin
  • the invention also provides various uses of a compound according to the invention, including for the preparation of a medical device, for diagnostic purposes, for treatment of a subject suspected to be suffering of an intoxication with a bacterial toxin such as an ABs toxin, or for treatment of a subject suspected to be suffering from an autoimmune disease such as an autoimmune neuropathy.
  • said medical device is a plasmapheresis unit, for example provided with a compound having multivalent binding capacity for a bacterial toxin, preferably an AB5 toxin such as Cholera toxin (CT), E. coli heat labile toxine (LT) or Shiga toxin (Stx), for example linked to a carrier, such as a monolith, according to the invention.
  • CT Cholera toxin
  • LT E. coli heat labile toxine
  • Stx Shiga toxin
  • the invention also provides a diagnostic device comprising for example a compound coupled or linked to a solid carrier, such as an affinity monolith according to the invention equipped for selective detection of antibodies and toxins, like SPR or ESI- MS, and use of a compound or composition according to the invention for the sensitive and fast diagnosis of autoimmune diseases and intoxications with bacterial toxins.
  • a diagnostic device comprising for example a compound coupled or linked to a solid carrier, such as an affinity monolith according to the invention equipped for selective detection of antibodies and toxins, like SPR or ESI- MS, and use of a compound or composition according to the invention for the sensitive and fast diagnosis of autoimmune diseases and intoxications with bacterial toxins.
  • Figure 4 Synthesis of the ligands. Eeagents and conditions: a) 1, CuSCU, sodium ascorbate, DMFZH 2 O, 8O 0 C, 20 min. yields: 57% for 4b, 78% for 5b, 76% for 6b. b) 3, CuSO 4 , sodium ascorbate, DMF/H2O, 8O 0 C, 20 min 64% for 5c, 37% for 6c.
  • Luminex beads detecting cholera toxin B-subunit and serum anti-GMl antibodies.
  • Luminex beads covalently coated with GMl-alkyl and detecting serum anti-GMl antibodies (from normal control NC 18 and GBS patient F 102A) and cholera toxin B-subunit.
  • FIG. 7 ELJSA detecting serum anti-GMl antibodies. ELISA plates coated non- covalently with GMl, and covalently with GMl-alkyl and GMl-dimers. Incubation with serum from 2 healthy controls (NC30, BCN23) and GBS patients (F120, F183).
  • FIG. 8 Specific depletion of serum anti-GMl antibodies with dendrimers. Depletion experiment with tetravalent GMl-dendrimers: no depletion of serum anti- GQIb antibodies (F297C), but depletion of serum anti-GMl antibodies (F102A).
  • FIG. 9 Depletion of serum anti-GM2 antibodies with monoliths coated with GM2 mimics. Immunoadsorption of serum with anti-GM2 antibodies: no depletion with blank monolithic column, but depletion with monolithic column coated with GM2 mimics.
  • FIG. 10 Binding of serum anti-GM2 antibodies to GM2-coated monolithic column.
  • Two monolithic columns were incubated with serum from a GBS patient with anti- GM2 antibodies, then washed and then incubated with FITC-conjugated anti-human immunoglubulin.
  • the monolithic columns were blank (A) or coated with a GM2 mimic (B). Observed fluorescence in column B clearly demonstrates homogenous binding of immunoglobulins from the serum to the column where column A demonstrates no binding.
  • the cholera toxin is a prime example of a multivalent protein and is capable of binding simultaneously to the carbohydrate moieties of five GMl gangliosides.
  • the binding of the five B subunits is of critical importance for the internalization and subsequent disease process of this ABe toxin.
  • the development of strong binding agents to the toxin is of interest for the development of disease prevention/treatment, but also for the detection of toxin in patient samples or in materials suspect of terrorist origin.
  • the cholera toxin serves as a benchmark case to test multivalent strategies. As such, several systems have been reported that showed varying degrees of multivalency effects.
  • a multivalent conjugated modified GMlos was prepared in very small quantities via reductive amination, and found to enhance binding up to 250-fold.
  • the invention provides strong inhibition of Cholera Toxin by multivalent GMl derivatives.
  • GMlos authentic GMl oligosaccharide sequence
  • GMlos conjugates 1 and 2 and related GM2os conjugate 3 were prepared on a 100 mg scale via a route containing chemical synthesis and enzymatic steps.
  • the CIl tail of 1 and 3 contains an azido group to enable its conjugation via "click" chemistry, a method which extends the range of organic and inorganic substrates to which oligosaccharides can be attached significantly.
  • the dendritic scaffolds 5a, 6a and the monovalent reference compound 4a were derived from our earlier developed dendrimers b that were functionalized with alkynes by coupling to 4-pentynoic acid.
  • an ELISA assay was used.
  • wells of a 96-well plate were coated with GMl ganglioside, and after blocking with BSA, horseradish peroxidase (HRP)-conjugated CTBe was allowed to bind to the surface with or without inhibitors.
  • HRP horseradish peroxidase
  • the monovalent GMlos derivative 2 exhibited an IC50 in the micromolar range (19 ⁇ M, Table 1). Since the GMlos oligosaccharide is reported to have a Kd of 43 nM, the high inhibitory concentration of the conjugated GMlos 2 indicates that the toxin binds strongly to the ELISA plate due to multivalent binding.
  • the prepared compounds exhibited unprecedented affinities, not only because the strongest known CT ligands (GMlos) were used, but also because these ligands were combined with the strongest multivalency effects observed for CT.
  • GMlos CT ligands
  • each of the GMlos moieties bound more than 20 thousand-fold stronger than monovalent 2. Possibly the multivalency effects could be even larger, since the detection limit of the assay appears to have been reached.
  • GM2os support this notion.
  • Monovalent GM2os was not measured because an expected low affinity indicated that it would take too much material to perform the assay.
  • divalent 5c was prepared and its inhibitory potency could just be observed with an IC50 of 2 mM.
  • compound 4 is poorly soluble in water: it forms a gel, and as such is not amenable for enzymatic modification.
  • Compound 4 was first dissolved in 100% MeOH. After addition of the different reaction components we obtained a mixture of methanol/water (20/80 v/v) which was compatible with CST-06, the sialyltransferase used to add NeuAc. The mixture of compound 4, sialyltransferase CST-06 and CMP- NeuAc was kept at 37 °C for 1 h, after which TLC analysis unambiguously showed that lactoside 4 was converted completely.
  • the target GM3-C ⁇ (5) was subsequently bound on a Sep-Pak column, which was then washed with ⁇ 2O to elute hydrophilic compounds (such as the buffer and the nucleotide) and finally the GM3-Cn was eluted with MeOH in a 92% yield.
  • GM2-C ⁇ (6) For synthesis of compound GM2-C ⁇ (6), we added the different components directly to the GM3-Cn (5) reaction mixture which resulted in a final concentration of 10% (v/v) methanol. Compound 5 was in this medium reacted with the in situ- generated UDP-GaINAc in a one-pot mixture containing UDP-GIcNAc, the UDP- GIcNAc 4'-epimerase (CPG- 13) and the ⁇ -l,4-iV-acetylgalactosaminyltransferase (CJL-30). The GM2-C ⁇ (6) was bound to a Sep-Pak column as described above for GM3-Cii (5) and eluted with MeOH.
  • CTBs inhibition assay A 96-well plate was coated with a solution of GMl (100 ⁇ L, 2 ⁇ g/mL) in PBS buffer. Unattached ganglioside was removed by washing with PBS and the remaining binding sites of the surface were blocked by BSA (1%) followed by PBS-washing. Samples of toxin-perosddase conjugate (CTB-HRP Sigma) and inhibitor in PBS with BSA (0.1%) and Tween 20 (0.05%) were incubated at r.t. for 2 h and were then transferred to the GMl-coated plate. After 30 min of incubation the solution was removed followed by washing steps with BSA (0.1%)/Tween 20 (0.05%) in PBS.
  • Monolithic carriers In recent years, monolithic (continuous bed) stationary phases have found wide application, e.g. in analytical columns, capillaries and channels of microfluidic chips. Affinity chromatographic methods using monolithic matrixes have been reported. It has been demonstrated that affinity chromatography using monolithic beds has a high potential for the analysis of biological macromolecules because the macroporous structure of the bed improves mass transport, allows a high mobile phase velocity without losing resolution and they are easily prepared. So far only few publications have appeared [Bedair, M. and El Rassi, Z., J. Chromatogr. A, 1044 (2004) 177-186; Bedair, M. and El Rassi, Z., Affinity chromatography with monolithic capillary columns - J.
  • Polystyrene standards (Mr 707, 1920, 3460, 5610, 12,500, 27,500, 51,500, 125,000, 271,000, 524,000, 864,000, 1,530,000) were from Polymer Standards Service (Mainz, Germany).
  • THF and benzene were HPLC grade solvents from Sigma (The Netherlands).
  • Reaction mixture 30 ⁇ L of HEMA, 20 mg of DATD, 12.5 mg Of(NHi) 2 SO 4 and 250 ⁇ L of PDA buffer solution (160 mg of PDA was dissolved in 2 mL of 50 mM sodium phosphate buffer, pH 7.0) were taken in an Eppendorf tube and mixed well. The reaction mixture was then degassed by vacuum for 5 min. 10 ⁇ L of 10% (w/v) aqueous AMPA solution was added to the above reaction mixture and mixed well. The resulting polymerization mixture was taken in a disposable syringe and injected during 5 min into the capillary by using a syringe pump at a flow rate of 1 ⁇ L/min.
  • fused-silica capillary (150, 180, 250 ⁇ m i.d) inner surface was performed as described in [Ericson, C, Liao, J. L., Nakazato, K, Hjerten, S., J. Chromatogr. A, 767, 1997, 33-41] with slight modification: acetone (20 min), distilled water (20 min), 0.1 M HCl (20 min), distilled water (20 min), 1 M NaOH (1 hr), distilled water (20 min) and acetone (20 min).
  • Gangliosides play a crucial role as target molecules for bacterial toxins and serum antibodies in a wide range of human infectious and auto-immune diseases. The specificity and pathogenic effects of these bacterial toxins and serum antibodies has been identified in most of these diseases.
  • the aimed toxins in this invention are cholera toxin binding to GMl in patients with cholera, enterotoxm binding to GMl in patient with travellers diarrhoea, diphtheria toxin binding to disialic gangliosides (including GDIb, GD2 and GD3) in patients with diphtheria, tetanus toxin binding to disialic gangliosides (including GDIb, GD2 and GD3) in patients with tetanus, and any other toxins binding to ganglioside or glycoconjugates in other patients with infectious diseases.
  • the invention provides treatment and diagnostic methods and means relating to human diseases associated with a ⁇ to-antibodies to gangliosides and other glycoeonjugates.
  • Most are post-infectious or autoimmune neural diseases, including Guillain-Barre syndrome (GBS) and all its variants, Miller Fisher syndrome (MFS), acute ophthalmoplegia, acute bulbar palsy, mononeuritis multiplex, (polyneuritis cranialis, acute diplegia facialis, acute brachealis palsy, acute autonomic neuropathy, acute sensory neuropathy, chronic inflammatory demyelinating neuropathy (CIDP), multifocal motor neuropathy (MMN), paraprotein-related polyneuropathy (PP-PNP), other forms of chronic ataxic neuropathy with ophthalmoplegia M-protein ataxia and disialic ganglioside antibodies to (CANOMAD), and all other forms of axonal or demyelinating immune-mediated neuropathies, acute ataxia (being cere
  • high binding oligosaccharide compositions in human diseases
  • the high binding oligosaccharide compounds and compositions can be used in the diagnosis, prevention and treatment of human diseases associated with toxins and antibodies binding to gangliosides and glycoconjugates.
  • the invention provides the detection of any toxin that specifically binds to oligosaccharide structures, binding oligosaccharides as such, glycolipids and glycoproteins.
  • the high binding oligosaccharide compounds and compositions have a high affinity for toxins, including the cholera toxin.
  • This toxin binds specifically to the ganglioside GMl and binding is crucial to exert its pathogenic effects leading to the clinical manifestations of cholera.
  • Luminex beads covalently coated with GMl-ClI alkyl specifically captured the cholera toxin B- subunit.
  • the compounds covalently bound to solid carriers such as beads, monolithic beds and ELISA plates can therefore be used as highly sensitive and highly specific assays to detect bacterial toxins.
  • solid carriers such as beads, monolithic beds and ELISA plates
  • These techniques can be used in the diagnosis or exclusion of these infectious diseases by demonstrating these toxins in serum, sputum and faeces samples from suspected patients. After diagnosis and treatment of these patients the changes in levels of these toxins can also be ⁇ ised to monitor and evaluate the effects of therapy.
  • the detection of antibodies to gangliosides using a compound or composition as provided herein has several applications in patients with related neurological diseases. Detection of these antibodies by the compositions can be used to (1) diagnose or exclude one of the aforementioned auto-antibody related diseases, (2) identify patients with a distinct pathogenic and/or clinical subtype of one of these diseases, (3) identify and predict in these patients a distinet type of clinical course (good or poor prognosis after certain types of treatment), (4) identify patients who require distinct forms of specific and/or additional treatments, and (5) monitor the effects of treatment based on the changes in antibody titre and specificity.
  • An example of these applications is the detection of IgG antibodies to the ganglioside GMl in the serum from patients with neuropathies. The presence of these antibodies in serum was found to be highly associated with (ad. 1) the diagnosis GBS, (ad. 2) a distinct pathogenic and clinical subtype of GBS caused by an antecedent
  • Luminex beads covalently coated with GMl-CIl alkyl bind with high sensitivity and specificity serum antibodies to GMl in a patient with GBS.
  • Serum from a healthy control without antibodies to GMl in routine ELISA was negative in the bead-based assay.
  • the ratio between this positive and negative serum was much higher in this assay than in routine GMl ELJSA, indicating that the signal-noise ratio in this assay is significantly better than in routine EUSA.
  • an ELISA type assay was used.
  • wells of a 96-well plate were coated with GMl ganglioside, and after blocking with BSA, horseradish peroxidase (HRP)-conjugated CTBs was allowed to bind to the surface in the presence or absence of inhibitors.
  • HRP horseradish peroxidase
  • the monovalent GMlos derivative 2 exhibited an ICBO in the micromolar range (19 ⁇ M, Table 1).
  • the divalent 5b was subsequently measured, and found to have an ICso that was almost 4 orders of magnitude lower, i.e 2.0 nM.
  • the tetravalent 6b gave an ICBO which was an additional order of magnitude lower (0.23 nM).
  • Antibodies to various gangliosides are highly neurotoxic and are considered to be the main cause of neurological deficits in the aforementioned diseases. For none of these diseases at present a specific form of treatment exists. Most of these patients are treated with apecific forms of treatment such as plasmapheresis and IVIg, which have only a modest therapeutic effect. Moreover, a major disadvantage of plasmapheresis is that it needs a skilled centre and facilities and may lead to severe cardiovascular complications. Major disadvantages of TVIg are the expenses, the shortage of material, and the possible contaminations in this biological product. The invention intends to deliver a new and rational form of treatment for these diseases. The invention can be applied in several ways to selectively neutralizes the pathogenic effects of the anti-ganglioside antibodies in these diseases.
  • the invention provides an highly effective and selective immunoadsorption because of (a) the use of authentic ganglioside oligosaccharide sequences as the most physiological and relevant target, (b) the use of an extremely high density of these oligosaccharide sequences required for multivalent binding and for the conformational epitopes mimicking those in the physiological clusters of gangliosides in lipid rafts at the neural membranes, (c) the use of multivalent dendrimeric scaffold further increasing the epitope density, (d) the use of elongated spacer arms and (e) the use of the monolithic composites resulting in a strong increase of the efficient surface for ant ⁇ body-ligand interaction.
  • the covalent binding of the synthetic ganglioside mimics to the monolith prevents the detachment of these mimics from the column during plasmapheresis and the contamination of plasma returned to the patient. Because of this covalent binding the columns may be reused after regeneration.
  • This experiment further demonstrates the highly selective immunoadsorption of anti-ganglioside antibodies from the serum of patients with autoimmune neuropathy.
  • monolithic beads prepared from grinding a polymerised monolithic mass, and containing synthetic ganglioside mimics, can be administered orally as a pharmaceutical composition to deplete anti- gangloside antibodies in the intestinal tract for the acute treatment of a subject suffering of an intoxication with a bacterial toxin, such as cholera toxin.
  • immunoadsorption columns are used containing beads covalently and densely coated with single or various synthetic ganglioside mimics which have similar advantages as the coated monoliths (although the effective surface for antibody-ligand interaction may be less than in coated monoliths).
  • Monolithic columns, containing synthetic ganglioside mimics according to the invention can be used for sensitive diagnostic purposes, for instance for the detection of GBS antibodies.
  • the monolithic affinity column should be connected to a surface plasmon resonance (SPR) device coated with ganglioside mimics or connected to an electrospray ionization mass spectrometer equipped with a nanospray interface capable of specifically detecting antibody proteins or toxins.
  • SPR surface plasmon resonance
  • serum or another biological fluid containing pathogenic antibodies for example IgM antibodies against GM2
  • the column is washed and then the antibodies are eluted with a suitable medium. The eluted antibodies are directed in high concentration and pure form to the SPE device.
  • SPE is a powerful technique to measure biomolecular interactions in real-time and in a label-free environment. While the ligand is immobilized on a gold sensor surface, the proteins are free in solution and passed over the surface. Association and dissociation on the gold surface with immobilised ligands can be measured sensitively and rapidly through minute differences in refractive index.
  • ESI-MS can be used as an alternative detection device. Such a combination of affinity chromatography and selective detection, enables fast and sensitive diagnosis of diseases at an early stage and replaces more elaborate ELJSA methods.

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Abstract

L'invention concerne des compositions d'oligosaccharide ayant une affinité pour des protéines et l'utilisation médicale ou biotechnologique de telles compositions. L'invention fournit une composition ayant une capacité de liaison polyvalente pour une protéine comprenant au moins deux fractions d'oligosaccharide liées à un dendrimère, ayant une capacité de liaison polyvalente pour une toxine bactérienne, de préférence une toxine AB5.
PCT/NL2008/050443 2007-07-02 2008-07-02 Composés d'oligosaccharide de liaison élevée, compositions et leurs utilisations WO2009005353A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998026662A1 (fr) * 1996-12-19 1998-06-25 The Penn State Research Foundation Composes et methodes pour traiter et prevenir les maladies bacteriennes et virales
DE19758105A1 (de) * 1997-12-18 1999-06-24 Schering Ag Dendritische Polymer-Saccharid-Konjugate, diese enthaltende pharmazeutische Mittel, Verfahren zu ihrer Herstellung und ihre Verwendung
US6310043B1 (en) * 1998-08-07 2001-10-30 Governors Of The University Of Alberta Treatment of bacterial infections
EP1733742A1 (fr) * 2005-06-17 2006-12-20 Universiteit Utrecht Holding B.V. Dendrimères substitués de façon multivalente par des groupes actifs

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998026662A1 (fr) * 1996-12-19 1998-06-25 The Penn State Research Foundation Composes et methodes pour traiter et prevenir les maladies bacteriennes et virales
DE19758105A1 (de) * 1997-12-18 1999-06-24 Schering Ag Dendritische Polymer-Saccharid-Konjugate, diese enthaltende pharmazeutische Mittel, Verfahren zu ihrer Herstellung und ihre Verwendung
US6310043B1 (en) * 1998-08-07 2001-10-30 Governors Of The University Of Alberta Treatment of bacterial infections
EP1733742A1 (fr) * 2005-06-17 2006-12-20 Universiteit Utrecht Holding B.V. Dendrimères substitués de façon multivalente par des groupes actifs

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BORGES A R ET AL: "DENDRIMERS AND ANTIVIRALS: A REVIEW", CURRENT DRUG TARGETS. INFECTIOUS DISORDERS, BENTHAM SCIENCE PUBLISHERS, HILVERSUM, NL, vol. 5, no. 3, 2005, pages 247 - 254, XP008058495, ISSN: 1568-0053 *
PATEL ET AL: "Attenuation of beta-amyloid induced toxicity by sialic acid-conjugated dendrimeric polymers", BIOCHIMICA ET BIOPHYSICA ACTA - GENERAL SUBJECTS, ELSEVIER SCIENCE PUBLISHERS, NL, vol. 1760, no. 12, 7 November 2006 (2006-11-07), pages 1802 - 1809, XP005851534, ISSN: 0304-4165 *
SAKAMOTO ET AL: "Thiosialoside clusters using carbosilane dendrimer core scaffolds as a new class of influenza neuraminidase inhibitors", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, OXFORD, GB, vol. 17, no. 3, 19 January 2007 (2007-01-19), pages 717 - 721, XP005835925, ISSN: 0960-894X *

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