WO2004065400A1 - Nouveaux epitopes de liaison a helicobacter pylori et utilisation desdits epitopes - Google Patents

Nouveaux epitopes de liaison a helicobacter pylori et utilisation desdits epitopes Download PDF

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Publication number
WO2004065400A1
WO2004065400A1 PCT/FI2004/000027 FI2004000027W WO2004065400A1 WO 2004065400 A1 WO2004065400 A1 WO 2004065400A1 FI 2004000027 W FI2004000027 W FI 2004000027W WO 2004065400 A1 WO2004065400 A1 WO 2004065400A1
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helicobacter pylori
nac
binding
oligosaccharide
glc
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PCT/FI2004/000027
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English (en)
Inventor
Halina Miller-Podraza
Susann Teneberg
Jonas ÅNGSTRÖM
Karl-Anders Karlsson
Jari Natunen
Jari Helin
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Glykos Finland Oy
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Priority claimed from PCT/FI2003/000039 external-priority patent/WO2003059924A1/fr
Application filed by Glykos Finland Oy filed Critical Glykos Finland Oy
Publication of WO2004065400A1 publication Critical patent/WO2004065400A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • 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
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • 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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical

Definitions

  • the present invention describes a substance or receptor binding to Helicobacter pylori, and use thereof in, e.g., pharmaceutical and nutritional compositions for the treatment of conditions due to the presence of Helicobacter pylori.
  • the invention is also directed to the use of the receptor for diagnostics of Helicobacter pylori.
  • Helicobacter pylori has been implicated in several diseases of the gastrointestinal tract including chronic gastritis, non-steroidal anti-inflammatory drug (NSAID) associated gastric disease, duodenal and gastric ulcers, gastric MALT lymphoma, and gastric adenocarcinoma (Axon, 1993; Blaser, 1992; DeCross and Marshall,
  • NSAID non-steroidal anti-inflammatory drug
  • Helicobacter pylori is also involved in totally or partially non-gastrointestinal diseases including sudden infant death syndrome (Kerr et al, 2000 and US 6,083,756), autommune diseases such as autoimmune gastritis and pernicious anaemia (Appelmelk et al, 1998; Chmiela et al, 1998; Clayes et al, 1998; Jassel et al, 1999; Steininger et al, 1998) and some skin diseases (Rebora et al, 1995), pancreatic disease (Correa et al, 1990), liver diseases including adenocarcinoma (Nilsson et al, 2000; Avenaud et al, 2000) and heart diseases such as atherosclerosis (Farsak et al, 2000).
  • sudden infant death syndrome Kerr et al, 2000 and US 6,083,756
  • autommune diseases such as autoimmune gastritis and pernicious anaemia (Appelmelk et al, 1998; Chmiela et al,
  • Glycoconjugates both lipid- and protein-based, have been reported to serve as receptors for the binding of this microorganism, e.g., sialylated glycoconjugates (Evans et al, 1988), sulfatide and GM3, NeuNAc ⁇ 3Lac ⁇ Cer, (Saitoh et al, 1991), Le b determinants, Fuc ⁇ 2Gal ⁇ 3(Fuc ⁇ 4)GlcNAc (Boren et al, 1993), polyglycosylceramides, heterogenous branched and linear fucosylated and sialylated stractures with sialic acid as part of receptor (Miller-Podraza et al, 1996; 1997a), lactosylceramide, Lac ⁇ Cer(hydroxyl fattyacids) (Angstrom et al, 1998) and gangliotetraosylceramide (Lingwood et al, 1992; Angstrom et al, 1998).
  • Helicobacter pylori include polysaccharide heparan sulphate, (GlcNAc ⁇ 4GlcA ⁇ /IdoA ⁇ 4) n comprising various sulphates, (Ascensio etal, 1993) as well as the phospholipidphosphatidylethanolamine (Lingwood et al, 1992). These sequences are different from the epitopes described by the present invention.
  • the polysaccharides chondroitin sulphate, chondroitin, and hyaluronic acid, and even non-specified fragmnets derived from these as polyvalent conjugates, have been also described as receptors for Helicobacter pylori (FI20011403).
  • FI20011403 does not describe minimum size of the oligosaccharide effective for inhibition or binding of H. pylori (if any possible in monovalent form) and does not give effective binding structure. Moreover, the exact sulphation status of the chondroitin oligosaccharides was not described nor possible natural variations of the oligosaccharide sequences or the role of these in specific binding to H pylori.
  • compositions comprising chondroitin sulphate, chondroitin sulphate or hyaluronic acid polysaccharides or fragments thereof (e.g. WO09827988, EP0704216, US5894070, US4524066,
  • WO9407505 in various therapheutic compositions.
  • WO9106303 claims chondroitin sulphate as molecules comprising chondroitin sulphate disaccharide, but specific oligosaccharides or non-sulphated oligosaccharides were not claimed. Chondroitin sulphate oligosaccharides has not been described as medicines and specific molecular structures has not been indicated.
  • Some applications describe hyaluronic acid oligosaccharides in medical applications (WO0204471, WO9957301), the latter application describes ester derivatives of hyaluronic acid.
  • WO03002128 describes the use of hyaluronic acid and chondroitin (sulfate) against H. pylori.
  • WO03002128 does not reveal the binding mode of H. pylori towards oligosaccharide sequences, actual specific oligosaccharide sequence based H. pylori, binding epitopes, especially effective terminal epitopes, or the size of the binding epitopes.
  • Gangliotetraosylceramide has sequence Gal ⁇ 3GalNAc ⁇ 4Gal ⁇ 4Glc ⁇ Cer.
  • An international application WO 02056893 claims similar terminal sequences Gal ⁇ 3GalNAc of gangliotetraosylceramide and disaccharide terminal Gal ⁇ 3GlcNAc of lactotetraosylceramide Gal ⁇ 3GlcNAc ⁇ 3Gal ⁇ 4Glc ⁇ Cer as H. pylori binding sequences.
  • the minimal disaccharide of the present invention GalNAc ⁇ 4Glc(A)o / ⁇ (NAc) 0 / ⁇ has different monosaccharides and different linkage configuration compared to Gal ⁇ 3GalNAc.
  • US patent Krivan et al. 5,446,681 (November 1995) describes bacterium receptor antibiotic conjugates comprising an asialo ganglioside coupled to a penicillin antibiotic. Especially is claimed the treatment of Helicobacter pylori with an amoxicillin-asialo-GMl conjugate.
  • the oligosaccharide sequences/glycolipids described by the invention do not belong to the ganglioseries of glycolipids.
  • the saccharide sequence GlcNAc ⁇ 3Gal has been described as a receptor for Streptococcus (Andersson et al, 1986). Some bacteria may have overlapping binding specificities, but it is not possible to predict the bindings of even closely related bacterial adhesins. In case of Helicobacter pylori the saccharide binding molecules, except the Lewis b binding protein are not known.
  • the present invention relates to a substance or receptor binding to Helicobacter pylori comprising the oligosaccharide sequence
  • ql,q2, rl, r2, r3, si and s2 are each independently 0 or 1, so that at least r2 or q2 is 1, at least si or s2 is 1, or Hex2 is Glc, with the proviso that when s2 is 0, then Hexl is Glc and preferably rl is 0; Hexl is galactose (Gal), glucose (Glc) or mannose (Man), Hex2 is Gal or Glc and analogs or derivatives of said oligosaccharide sequence for binding or inhibiting Helicobacter pylori.
  • the obj ects of the invention are the use of the Helicobacter pylori binding oligosaccharide sequences described in the invention as a medicament, and the use of the same for the manufacture of a pharmaceutical composition, particularly for the treatment of any condition due to the presence of Helicobacter pylori in a subject.
  • the present invention also relates to the methods for the treatment of conditions due to the presence of Helicobacter pylori.
  • the invention is also directed to the use of the receptor(s) described in the invention as Helicobacter pylori binding or inhibiting substance for diagnostics of Helicobacter pylori.
  • Another object of the invention is to provide substances, pharmaceutical compositions and nutritional additives or compositions containing Helicobacter pylori binding oligosaccharide sequence(s).
  • Yet another object of the invention is the use of the above-mentioned Helicobacter pylori binding substances for the production of a vaccine against Helicobacter pylori.
  • Figs. 1 A and IB Thin-layer chromatogram with separated glycosphingolipids detected with 4-methoxybenzaldehyde (1 A) and autoradiogram after binding of radiolabeled Helicobacter pylori strain 032 (IB).
  • the glycosphingolipids were separated on aluminum-backed silica gel 60 HPTLC plates (Merck) using chloroform/methanol/water 60:35:8 (by volume) as solvent system.
  • the binding assay was done as described in the "Materials and methods" section.
  • the lanes contained: lane 1) Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4Glc ⁇ lCer (neolactotetraosylceramide), 4 ⁇ g; lane 2) Gal ⁇ 3Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4Glc ⁇ lCer (B5 glycosphingolipid), 4 ⁇ g; lane 3) Gal ⁇ 3Gal ⁇ 4GlcNH 2 ⁇ 3Gal ⁇ 4Glc ⁇ lCer, 4 ⁇ g; lane 4) Gal ⁇ 3(Fuc ⁇ 2)Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4Glc ⁇ 1 Cer (B6 type 2 glycosphingolipid), 4 ⁇ g; lane 5) GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4Glc ⁇ lCer, 4 ⁇ g; 6) Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇
  • glycosphingolipids 4 ⁇ g.
  • the sources of the glycosphingolipids are the same as given in Table 1.
  • GlcA ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc A sample of the product was deposited on a DHB matrix and analyzed in the reflector positive ion mode.
  • Fig. 3 MALDI-TOF mass spectrum of the purified amidation product of chondroitin tetrasaccharide (Glc[ANH 2 ] ⁇ 3GalNAc ⁇ 4Glc[A-NH 2 ] ⁇ 3GalNAc): m/z 797.2 ([M+Na] + ) and 813.2 ([M+K] + ).
  • A-NH 2 indicates the glucuronamides -CO-NH 2
  • Fig.6 MALDI-TOF mass spectrum of the purified DAP- 16 dendrimer conjugate of Glc[ANH 2 ] ⁇ l,3GalNAc ⁇ l,4Glc[ANH 2 ] ⁇ l,3GalNAc.
  • the broad signal around 12000-17000 Da represents a nonspecific dimer, i.e. [2M+H] + , of the dendrimeric conjugate.
  • Fig.7 MALDI-TOF mass spectrum of the reduced hyaluronic acid oligosaccharide mixture. See text for peak details.
  • WO 02056893 showed optimal trisaccharide binding sequences for Helicobacter pylori having the oligosaccharide sequence Gal(A) q ⁇ (NAc) r ⁇ /Glc(A) q2 (NAc) r2 ⁇ 3/ ⁇ 3Gal ⁇ 4Glc(NAc) u wherein ql, q2, rl, r2, and u are each independently 0 or 1, and wherein lactose based sequences are present in polyvalent form or in high concentration. Also non- reducing-end elongated variants were shown to be effective. Moreover, the studies showed that the oligosaccharide sequence can vary in many positions without losing its H. pylori binding activity.
  • the present invention allows adding specific modifications to the sequence adding possible modifications of the reducing end side monosaccharide residue and additional variations to the middle and reducing end monosaccharide residues. Furthermore, the present invention represents novel derivatives of uronic acids and method for their preparation. Moreover the present invention is directed to the use of the oligosaccharide backbone in screening assays, in design of combinatorial chemistry libraries and in molecular modeling for design of better analogues or derivatives of the oligosaccharide sequences.
  • the present invention shows that Gal ⁇ 4 residue can be replaced in the oligosaccharide sequences by GalNAc ⁇ 4.
  • the examples show that GalNAc supports the H. pylori binding activity of lacdiNAc type oligosaccharide sequences such as GalNAc ⁇ 4GlcNAc, and chondroitin oligosaccharide sequeces Glc(A-N ⁇ 2 ) ⁇ 3GalNAc ⁇ 4Glc(A-N ⁇ 2 ) ⁇ 3GalNAc, wherein A-NH 2 indicates the glucuronamides, and GlcA ⁇ 3GalNAc ⁇ 4GlcA ⁇ 3GalNAc ⁇ 4GlcA ⁇ 3GalNAc.
  • the chondroitin structures show the usefulness of the uronic acid also in the middle or at the reducing end of the oligosaccharide chain.
  • the examples further include a library of uronic acid amide derivatives, from methyl amine to octadecylamine, from which smaller structures were found to be more active than larger structures.
  • the novel binding structures share a common conformation. The putative conformation indicates that there are possible areas of the molecules which can be modified such as position number two of Gal/GalNAc ⁇ 4 and reducing end Glc/GlcA/GlcNAc, which both obviously tolerate amine derivative group and hydroxylstructures.
  • the present invention further discloses that hyaluronic acid oligosaccharides with similar sequences as chondroitin oligosaccharides, but containing GlcNAc residue in the place of GalNAc do also bind to Helicobacter pylori. Therefore a general formula of H. pylori binding saccharide epitopes can be drawn as Formula A.
  • the present invention provides a Helicobacter pylori binding substance comprising an oligosaccharide sequence according to Formula A
  • ql,q2, rl, r2, r3, si and s2 are each independently 0 or 1, so that at least r2 or q2 is 1, at least si or s2 is 1, or Hex2 is Glc, with the proviso that when s2 is 0, then Hexl is Glc and preferably rl is 0; and Hexl is galactose (Gal), glucose (Glc) or mannose (Man), Hex2 is Gal or Glc; and analogs or derivatives of said oligosaccharide sequence having binding activity to Helicobacter pylori for the prophylaxis or treatment of any condition due to the presence of Helicobacter pylori in a subject.
  • the present invention further provides a Helicobacter pylori binding substance comprising oligosaccharide sequence according to Formula B
  • ql,q2, rl, r2, r3, and si are each independently 0 or 1, so that at least r2 or q2 is 1; and Hexl is galactose (Gal), glucose (Glc) or mannose (Man), Hex2 is Gal or Glc.
  • trisaccharide epitopes are especially preferred, most preferably the trisaccharide epitope is a terminal oligosaccharide sequences.
  • the terminal monosaccharide unit is preferably ⁇ 3-linked as in chondroitin, chondroitin sulphate, hyaluronic acid or previously described N-acetyllactosamine oligosaccharide sequences.
  • Large scale cost effective availability of these materials makes these very useful and preferred raw materials for production of the oligosaccharide sequences according to the invention.
  • the present invention is thus further preferably directed to the Helicobacter pylori binding substance comprising oligosaccharide sequence according to Formula C
  • ql,q2, r2, and si are each independently 0 or 1, so that at least r2 or q2 is 1; and Hex2 is Gal or Glc.
  • Hyaluronic acid and chondroitin are preferred raw material for production of the oligosaccharide sequences according to the invention.
  • the present invention is further directed to N-acetylhexosamine based sequences derived from chondroitin or hyaluronic acid. More preferred embodiment includes thus Helicobacter pylori binding substance comprising oligosaccharide sequence according to the Formula D
  • chondroitin and hyaluronic acid oligosaccharide sequences are shown in Formulas E and F.
  • the formulas include carboxylic acid derivatives of the oligosaccharide sequences, especially amides and esters as described by the invention and partially or totally carboxylic acid reduced analogs thereof.
  • the reducing end of the oligosaccharide sequence is reduced, especially preferred, when the oligosaccharide sequence is a carboxylic acid reduced oligosaccharide sequence or oligosaccharide.
  • the oligosaccharide sequences are in a preferred embodiment sulphated, or in another preferred embodiment partially suphated chondroitin oligosaccharides.
  • the sulphated oligosaccharide sequence is sulphated so that one sulphate group is linked by natural linkage type and position to the GalNAc residue or residues of the oligosaccharide sequence.
  • the present invention is preferably directed to a Helicobacter pylori binding substance comprising an oligosaccharide sequence according to Formula E
  • ql,q2, and si are each independently 0 or 1.
  • the present invention is also preferably directed to a Helicobacter pylori binding substance comprising an oligosaccharide sequence according to Formula F
  • ql,q2, and si are each independently 0 or 1.
  • the present invention is further directed to novel disaccharide subepitope structures which contributes to the H. pylori binding.
  • the subepitopes may be used according to the invention, but in some case high concentration or polyvalent presentation is needed.
  • the subepitopes are preferably disaccharide sequences according to the Formula A.
  • the present invention is preferably directed to the use of the subepitope structures for design and/or synthesis of molecular libraries and analogs and/or derivatives of the oligosaccharides according to the invention.
  • a natural or enzymatically synthesized, disaccharide sequence subepitope is used for synthesis of an oligosaccharide sequence according to the invention, and in another embodiment for the synthesis of an analogue or derivative of an oligosaccharide sequence according to the invention.
  • More preferred disaccharide subepitopes include the following subepitope oligosaccharide sequences
  • Glc ⁇ 3GalNAc GlcA ⁇ 3GalNAc, Glc ⁇ 3Glc,
  • Glc ⁇ 3GlcNAc and GlcA ⁇ 3GlcNAc.
  • preferred disaccharide subepitopes include saccharides comprising GlcANAc in place of GlcA in any preferred sequences above.
  • the present invention also shows that for the optimal binding interaction the reducing end monosaccharide of the trisaccharide epitope should be in beta configuration.
  • the optimal presentation models can be used with similar reducing end structures for saccharide sequences according to Formulas A-F.
  • An example shows strong binding to reductively aminated GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc, while corresponding alpha anomer GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc was inactive or very weakly active under the experimental conditions. High binding activity was observed when the reducing end monosaccharide is Gal or GlcNAc.
  • GalNAc and Glc are preferred homologs having the same modifications.
  • the preferred specificity for ⁇ 6-type reducing end terminal was thus determined to be ⁇ 6Hex(Nac) 0 / ⁇ , wherein Hex is preferably Gal or Glc.
  • the reducing end monosaccharide residue is in open chain derivative form.
  • Preferred open chain forms of the reducing end of the substance include
  • reduced conjugated reducing end monosaccharide for example reductively animated reducing end
  • open chain conjugated reducing end such as an oxime of an aminooxy structure, NH 2 -O-R where R is the rest of the conjugation agent.
  • Reduced reducing ends are in aqueous environment balanced in alpha, beta and open chain forms.
  • the oxime of aminooxy acetic acid is usually in alpha, beta, and open chain forms in aqueous solutions.
  • the present invention is especially directed to favourable ⁇ 6-linked structures at the reducing end which are cost effective to produce and increase the binding to H. pylori.
  • the disaccharide GlcNAc ⁇ Gal can be produced effextively by organic synthesis or by enzymatic means and the substrate monosaccharides are relatively cheap compared to GalNAc.
  • GlcNAc ⁇ 6GlcNAc can be synthesized even more effectively chemically by traditional methods or by acid reversion chemistry. A suitable bacterial transferase for the synthesis of the substance is known.
  • the present invention is directed to the following disaccharide to pentasaccharide oligosaccharide sequences for the treatment of Helicobacter pylori.
  • the present invention is further directed to the oligosaccharides as Helicobacter pylori binding substances for treatment of infectious disease, preferably H. pylori dependent diseases.
  • the present invention is directed to the chemically modified or analogous oligosaccharide sequences to obtain lower biological degradation and higher or more specific activity towards H. pylori and/or other pathogens. Especially analogs comprising glucuronylamide are preferred.
  • the present invention is separately directed to the sequences according to the invention for use as medicine, preferably at least trisaccharide sequences are used.
  • the present invention is further directed to pharmaceutical composition comprising terminal oligosaccharide sequences according to the invention.
  • the structural element A indicates the presence or absence of uronic acid form of a monosaccharide residue, furthermore A indicates the presence of derivatized uronic acid, preferably as amides of ammonia, -CO-NH2 or organic amines of the carboxylic acid group at the 6 position.
  • Organic amides include preferably alkylamides, cycloalkylamides, and arylamides, including preferably amides from C1-C22 amines, more preferably C 1 -C6 amines, even more preferably from C 1 -C3 amines and most preferably amides from methylamine or ethylamine.
  • alkylamines of various sizes are aliphatic 1 -alkylamines.
  • Preferred arylamide has benzoic acid like benzyl-ring structure, preferred alkylamides include linear 1 -amino alkanes.
  • the amides of ammonia or methylamine or ethylamine, and amide from ammonia are more preferred when the molecule comprises uronic acid derivative other than non-reducing end uronic acid derivative or when the same oligosaccharide sequence comprises several uronic acid derivatives.
  • Other uronic acid derivatives include esters.
  • the preferred structures of the novel Helicobacter pylori binding oligosaccharide sequences comprise the terminal trisaccharide or disaccharide structures according to the Formula la
  • ql,q2, rl, r2, r3. r5 and s, and w are each independently 0 or 1, and Hexl, and Hex2 is a hexose structures, preferably galactose (Gal) or glucose (Glc) or mannose (Man), most preferably Gal or Glc, which may be further modified by the A and/or NAC groups; y is either alpha or beta indicating the anomeric structure of the terminal monosaccharide residue with the provisions that when w is 0, then at least one of the integers r2, ql(when s is also 1), or q2 is 1, preferably at least r2 or q2 is 1 and analogs or derivatives of said oligosaccharide sequence having binding activity to Helicobacter pylori for binding or inhibiting Helicobacter pylori.
  • Brackets [ ] or ⁇ ⁇ indicate herein that the structure is either present or absent, ( ) indicates presence or absence of a mono
  • the reducing end monosaccharide is ⁇ -linked, when w is 0.
  • the reducing end monosaccharide structure is an open chain reducing end derivative of the monosaccharide unit.
  • the structure is not linked to a ceramide, more preferably the structure is not linked to a ceramide comprising a hydroxyl fatty acid.
  • w is 1 and the reducing end monosaccharide is not glycosidically conjugated to another monosaccharide unit, more preferably the reducing end monosaccharide unit is in an open chain reducing end derivative.
  • a in the Formula is amide, methylamide or ethylamide of the carboxylic acid group of the glucuronic acid residue.
  • ql,q2,q3, rl, r2, r3,r5, r6, s, t, and u are each independently 0 or 1
  • Hexl, and Hex3 are hexose structures, preferably mannose (Man), galactose (Gal) or glucose (Glc), and most preferably Gal or Glc, which may be further modified by A and/or NAc groups
  • y is either alpha or beta indicating the anomeric structure of the terminal monosaccharide residue with the provisions that at least one of the integers r2, q2, or q3 is 1 or r5 is 1 when u is 1, preferably r2 is 1 or q2 is 1, and analogs or derivatives of said oligosaccharide sequence having binding activity to Helicobacter pylori for binding or inhibiting Helicobacter pylori.
  • the reducing end monosaccharide structure can be a conjugated derivative of the monosaccharide residue indicated by the formula, preferably an open chain reducing end derivative of the monosaccharide unit.
  • the reducing end monosaccharide structure is an open chain reducing end derivative of the monosaccharide unit.
  • s is 1.
  • Helicobacter pylori comprising the oligosaccharide sequence according to separately preferred formulas:
  • Hexl, Hex2 and Hex3 are hexose structures, preferably mannose (Man), galactose (Gal) or glucose (Glc), and most preferably Gal or Glc, which may be further modified by A and/or NAC groups;
  • B is a branch structure Hex2(NAc) r ⁇ 3, which can be present or absent, v is 0 or v is 1, only when the structure indicated by t is present (t is 1) in ⁇ 6-linked form;
  • the reducing end monosaccharide structure can be a conjugated derivative of the monosaccharide residue indicated by the formula, preferably an open chain reducing end derivative of the monosaccharide unit.
  • the reducing end monosaccharide structure is an open chain reducing end derivative of the monosaccharide unit.
  • s is 1.
  • a in the above oligosaccharide sequences indicates uronic acid of the monosaccharide residue or carbon 6 derivative of the monosaccharide residue, most preferably the derivative of carbon 6 is an amide of the uronic acid.
  • GalNAc ⁇ 4GlcNAc LacdiNAc
  • GalNAc ⁇ 4Glc reduced chondroitin type structures
  • oligosaccharide sequences are among the preferable Helicobacter pylori binding substances for the uses of the invention.
  • GalNAc ⁇ 3GalNAc ⁇ 4GlcNAc GalNAc ⁇ 3GalNAc ⁇ 4GlcNAc, GalNAc ⁇ 3GalNAc ⁇ 4GlcNAc, GlcNAc ⁇ 3GalNAc ⁇ 4GlcNAc.
  • Gal ⁇ 3GalNAc ⁇ 4GlcNAc Gal ⁇ 3GalNAc ⁇ 4GlcNAc
  • Glc ⁇ 3GalNAc ⁇ 4GlcNAc Glc ⁇ 3GalNAc ⁇ 4GlcNAc
  • Glc ⁇ 3GalNAc ⁇ 4GlcNAc GlcA ⁇ 3GalNAc ⁇ 4GlcNAc
  • GlcA ⁇ 3GalNAc ⁇ 4GlcNAc GlcA ⁇ 3GalNAc ⁇ 4GlcNAc
  • GalNAc ⁇ 4GlcNAcNAc GalNAc ⁇ 4GlcNAc
  • Preferred carboxylic acid reduced chondroitin type oligosaccharides include
  • s, t and u are integers so that s is either 0 or 1 and u is either 0 or 1 and t has any value or values (in a mixture) from 1 to 10.
  • t has any value or values from 1 to 5, more preferably t has values from 1 to 4 and most preferably t has values from 1 to 3.
  • the present invention is directed to the substances as medicines, preferred medicines include substance according to the above structure when si.
  • Glucuronic acids of natural type chondroitin oligosaccharide sequences can be reduced from carbodiimide conjugate or from methylesters by methods known in the art (see for example WO 0123398).
  • Preferred substances includes carboxylic acid reduced forms of preferred chondroitin oligosaccharides described below, for example
  • the present invention is directed to optimal receptor size glucuronic acid reduced chondroitin oligosaccharides as substances.
  • the preferred reduced oligosaccharide substance contains at least four, more preferably at least five monosaccharide residues. In another embodiment the preferred oligosaccharide has less than 11 monosaccharide residues, more preferably less than nine monosaccharide residues.
  • the present invention is further directed to composition comprising mixtures of the preferred oligosaccharides, which may further comprise smaller similar oligosaccharides. Preferably the mixtures comprise at least 20 %, more preferably at least 50 % and most preferably at least 80 % and even more preferably at least 90 % tetra- to octasacharides.
  • Preferred natural chondroitin type oligosaccharide sequences The present invention is especially directed to specific natural type chondroitin oligosaccharide sequences according to Formula 8
  • s, t and u are integers so that s is either 0 or 1 and u is either 0 or 1 and t has any value or values (in a mixture) from 1 to 10.
  • t has any value or values from 1 to 5, more preferably t has values from 1 to 4 and most preferably t has values from 1 to 3.
  • GlcA in the formula indicates glucuronic acid not derivatized at carboxylic acid.
  • the non-derivatized oligosaccharide sequences or oligosaccharides are especially preferred for various uses. These are derivatives of natural structures and are therefore especially preferred for functional foods, food additives, nutritional additives, and animal feeds.
  • the present invention is specifically directed to chondroitin oligosaccharides.
  • the chondroitin type oligosaccharide sequence is for use as a medicine for infectious diseases. Chondroitin oligosaccharides is considered also useful for therapheutical uses in foods, feeds, nutrients, self medication products, nutritional additives, food additives, and/or feed additives.
  • GlcA in the formula and specific structures below represent glucuronic acid which is derivatized to an amide
  • preferred amides includes amides formed from carboxylic acid group and ammonium/ammonia forming structure -CONH 2 , organic amides including alkylamides, cycloalkylamides, arylamides, including preferably amides from C1-C22 amines, more preferably C1-C6 amines, even more preferably from C1-C3 amines and most preferably amides from methylamine or ethylamine.
  • Preferred arylamide has benzoic acid like benzyl-ring structure.
  • Preferred alkylamides include linear 1 -amino alkanes.
  • chondroitin oligosaccharide sequences include oligosaccharides with non-reducing end terminal GlcA due to their high affinity towards H. pylori:
  • GlcA ⁇ 3GalNAc ⁇ 4GlcA GlcA ⁇ 3GalNAc ⁇ 4GlcA ⁇ 3GalNAc ⁇ 4GlcA
  • GlcA ⁇ 3GalNAc ⁇ 4GlcA ⁇ 3GalNAc ⁇ 4GlcA ⁇ 3GalNAc ⁇ 4GlcA GlcA ⁇ 3GalNAc ⁇ 4GlcA ⁇ 3GalNAc ⁇ 4GlcA ⁇ 3GalNAc ⁇ 4GlcA ⁇ 3GalNAc ⁇ 4GlcA ⁇ 3GalNAc ⁇ 4GlcA ⁇ 3GalNAc ⁇ 4GlcA
  • a specifically preferred group of oligosaccharide sequences includes structures comprising non-reducing-end terminal GlcA and reducing end terminal GalNAc.
  • Such oligosaccharides can be produced effectively from natural chondroitin sulphates by desulfation and hyaluronidase digestion (or by hyalurondase digestion and desulphation:
  • Yet another preferred embodiment includes oligosaccharide structures, when s is 0, contains terminal GalNAc:
  • GalNAc ⁇ 4GlcA GalNAc ⁇ 4GlcA ⁇ 3GalNAc ⁇ 4GlcA,
  • GalNAc ⁇ 4GlcA ⁇ 3GalNAc GalNAc ⁇ 4GlcA ⁇ 3GalNAc ⁇ 4GlcA ⁇ 3GalNAc, and
  • the present invention is in its preferred embodiments directed to the natural type oligosaccharide sequences such as chondroitin oligosaccharides as practically pure single components or as mixtures comprising 2-5 major component comprising more than 90 % of the oligosaccaride mixture.
  • the chondroitin sulphate oligosaccharides are preferably devoid of sulphate ester modifications.
  • the invention is further directed to analogous H pylori binding oligosaccharide sequences which have non- reducing terminal delta uronic acid residue (double bond between C4 and C5), which can be created by lyase type enzymes cleaving the corresponding polysaccharide.
  • Analogous hyaluronic acid oligosaccharide sequences Previously it was demonstrated that H. pylori binds to hyaluronic acid polysaccahride,Present invention allows to determine the binding active oligosaccharide epitopes.
  • the present invention is directed to the hyaluronic acid sequences for preparation medicines for the treatment of infectious diseases, especially due to the presence of H. pylori.
  • the preferred oligosaccharide sequences may also have the structure according to Formula 9b
  • s, t and u are integers so that s is either 0 or 1 and u is either 0 or 1 and t has any value or values (in a mixture) from 1 to 10.
  • t has any value or values from 1 to 5, more preferably t has values from 1 to 4 and most preferably t has values from 1 to 3.
  • the invention is further directed to analogous H. pylori binding oligosaccharide sequences which have non-reducing terminal delta uronic acid residue (double bond between C4 and C5), which can be created by lyase type enzymes cleaving the corresponding polysaccharide.
  • the invention is further directed to H pylori binding analog substances and screening higher affinity variants from the analogs based on formulas 1-8 wherein Gal is replaced by Glc.
  • the present invention is also directed to the hyaluronic acid derivative substances wherein A is amide as described for other oligosaccharide sequences according to the invention, preferably the glucuronic acid is derivatized to amides of ammonia, methylamine, or ethylamine.
  • the invention is further directed to the substances as medicines.
  • the present invention is directed to optimal receptor size glucuronic acid reduced hyaluronic acid oligosaccharides as substances.
  • the preferred reduced oligosaccharide substance contains at least four, more preferably at least five monosaccharide residues. In another embodiment the preferred oligosaccharide has less than 11 monosaccharide residues, more preferably less than nine monosaccharide residues.
  • the present invention is further directed to composition comprising mixtures of the preferred oligosaccharides, which may further comprise smaller similar oligosaccharides.
  • the mixtures comprise at least 20 %, more preferably at least 50 % and most preferably at least 80 % and even more preferably at least 90 % tetra- to octasacharides.
  • the present invention is further directed to a method producing chondroitin oligosaccharides from chondroitin sulphates.
  • the method steps involve 1. Removal of sulphates from chondroitin sulphate by chemical hydrolysis. There are several useful methods in the art for example incubating at 80-100 degrees of Celsius in dimethylsulfoxide in the presence of methanol or water. 2. Specifically hydrolyzing ⁇ 4-glycosidic bonds between GalNAc (GlcNAc) and GlcA.
  • the specific hydrolysis is preferably performed by acid hydrolysis, more preferably by a strong carboxylix acid, more preferably the strong carboxylic acid is trifluoroacetic acid.
  • the present invention is directed to specific hydrolysis of hyaluronic acid to oligosaccharides according to step 2.
  • the method steps can be performed in any order.
  • step 1 is performed first.
  • the purification of the product oligosaccharides involves anion exchange chromatography for the separation of the oligosaccharides. More preferably the purification involves anion exchange and gel filtration chromatography (size exclusion chromatography).
  • oligosaccharides are from 3-mers to 14-mers, more preferably 4-mers to 10-mers.
  • the present invention is further directed to methods for the production of amidated glucuronic acid comprising oligosaccharides and monosaccharides from glucuronic acid comprising polysaccharides.
  • the preferred polysaccharides includes pectin, desulphated chondroitin sulphate, and hyaluronic acid as well as bacterial exopolysaccharide comprising glucuronic acid.
  • 6-hydroxyls of a polysaccharide can be specifically oxidized to carboxylic acid groups.
  • the method involves following steps:
  • Amidation of glucuronic acid residues of the glucuronic acid comprising polysaccharide.
  • the amidation is performed from the polysaccharide activated by uronium type amide bond synthesis activator.
  • the carboxylic acid is activated by methyl ester.
  • the fragments are either oligosaccharides or monosaccharides.
  • chondroitin or hyaluronic acid polysaccharide is amidated and then hydrolyzed to oligosaccharides according to the invention.
  • hydrolysis is performed by a carboxylic acid which is more acidic than acetic acid, preferably by trifluoroacetic acid.
  • oligosaccharides are from 3-mers to 14-mers, more preferably 4-mers to 10-mers.
  • Present invention is further directed to novel substances which are useful for multiple therapheutical and other uses:
  • ql,q2, rl, r2, r3, r5 and s, and w are each independently 0 or 1, and Hexl, and Hex2 is a hexose structures, preferably galactose (Gal) or glucose (Glc), which may be further modified by the A and/or NAC groups;
  • y is either alpha or beta indicating the anomeric structure of the terminal monosaccharide residue with the provisions that when s is 0, q2 is 1 and A indicates an amide, when s is 1 both, ql, q2 are 1 with the provision that the molecule does not comprise two ⁇ - linked glucuronic acid units, or ql is 1 and q2 is 0 then y is alpha or r2 is 1, preferably A indicates glucuronic acid amide, methylamide or ethylamide.
  • the present invention describes a family of specific oligosaccharide sequences binding to Helicobacter pylori.
  • the structures of the glycosphingolipids used were characterized by proton NMR and mass spectrometric experiments. Numerous naturally occuring glycosphingolipids have been previously screened (in FI 20010118) by thin-layer overlay assay (Table 1).
  • the terms “analog” and “derivative” are defined as follows. According to the present invention it is possible to design structural analogs or derivatives of the Helicobacter pylori binding oligosaccharide sequences. Thus, the invention is also directed to the structural analogs and derivatives of the substances according to the invention.
  • the structural analogs according to the invention are molecules different from original oligosaccharide stractures studied and comprise the structural elements important for the binding of Helicobacter pylori to the oligosaccharide sequences.
  • the analogue is such molecule which can not be produced by chemical derivatization reaction in a few steps from the original oligosaccharide backbone while derivatives can be produced by chemical manipulation of the oligosaccharide stractures of the invention, derivative can be naturally produced also using derivatized monosaccharide building blocks or by other means.
  • derivatives can be easily produced from amino groups after de-N-acetylation of one or several N-acetylgroups of the molecule.
  • Derivatives can also be effectively produced from carboxylic acids groups of uronic acids and even hydroxyl groups can be derivatized for example by ether or ester groups.
  • the present invention is also specifically directed to the design of structural analogs and derivatives for the Helicobacter pylori binding oligosaccharide sequences.
  • design of effective structural analogs and derivatives it is important to know the structural element essential for the binding between Helicobacter pylori and the saccharides.
  • the important structural elements are preferably not modified or these are modified by very close mimetic of the important structural element. These elements preferably include the 4-, and 6-hydroxyl groups of the Gal ⁇ 4 residue in the trisaccharide and disaccharide epitopes. Also the positioning of the linkages between the ring stractures is an important structural element.
  • Acetamido group or acetamido mimicking group is preferred in the position corresponding to the acetamido group of the reducing end-GlcNAc of the di- or trisaccharide epitopes.
  • Acetamido group mimicking group may be another amide, such as alkylamido, arylamido, secondary amine, preferentially N-ethyl or N- methyl, O-acetyl, or O-alkyl for example O-ethyl or O-methyl.
  • amide derivatives from carboxylic acid group of the terminal uronic acid and analogues thereof are preferred.
  • the activity of non-modified uronic acid is considered to rise in lower pH.
  • the structural derivatives according to the invention are oligosaccharide sequences according to the invention modified chemically so that the binding to the Helicobacter pylori is retained or increased. According to the invention it is preferred to derivatize one or several of the hydroxyl or acetamido groups of the oligosaccharide sequences.
  • the invention describes several positions of the molecules which could be changed when preparing the analogs or the derivatives.
  • the hydroxyl or acetamido groups which tolerate at least certain modifications are indicated by R-groups in Formula 9.
  • the present invention is in a preferred embodiment directed for the production of analogues or derivatives according to the Formula 9.
  • analogues are tested for binding or inhibition of Helicobacter pylori and best binding sequences are selected for development of a product.
  • the molecules according to the invention or analogues or derivatives are tested for binding of other microbes or virases, preferably for binding to toxin A of Clostridium difficile.
  • Gal ⁇ 4GlcNAc and on C3-hydroxyl of the non-reducing terminal monosaccharide of the minimal trisaccharide epitopes Preferably, the analogues designed do not comprise substituents or bulky substituents at these positions.
  • oligosaccharide analogs for the binding of a lectin are well known.
  • numerous analogs of sialyl-Lewis x oligosaccharide has been produced, representing the active functional groups different scaffold, see page 12090, Sears and Wong 1996.
  • Similarily analogs of heparin oligosaccharides has been produced by Sanof ⁇ corporation and sialic acid mimicking inhibitors for the sialidase enzyme, such as accepted medical sialidase inhibitors against influenza by Hofmann-La Roche or Glaxo-Wellcome, by numerous groups.
  • the present invention is specifically directed to the design of analogues of the oligosaccharide structures according to the invention comprising ring stractures analogous to the monosaccharide residues of the oligosaccharide residues. More preferably the analogues are tested for binding or inhibition of Helicobacter pylori and best binding sequences are selected for development of a product. In another embodiment the molecules according to the invention or analogues or derivatives are tested for binding of other microbes or viruses, preferably for binding to toxin A of Clostridium difficile.
  • the oligosaccharide analog is build on a molecule comprising at least one six- or five-membered ring structure, more preferably the analog contains at least two ring stractures comprising 6 or 5 atoms.
  • a preferred analogue type of the oligosaccharide comprise a terminal uronic acid amide or analogue or derivative thereof linked to Gal/GalNAc ⁇ 4GlcNAc-saccharide mimicking structure.
  • 2 and 4 hydroxyl groups of the terminal monosaccharide residue are not important for binding and 6-hydroxyl can be modified to structures actually increasing the affinity of the molecule, high affinity analogs can be produced when these positions are modified.
  • the data shows that it is possible to design analogs which do not comprise all hydroxyl groups of the terminal monosaccharide residue.
  • the present invention is specifically directed to attaching various organic derivatization molecule such as aromatic or aliphatic cyclic organic residue for the 3 -position of terminal Gal/GalNAc production functional analog design.
  • the derivatization may be produced by special linker chemistry allowing linking the cyclic organic residues to 3-position of
  • the geometry and even length of linking stractures may be different from glycosidic bond stractures provided that the cyclic organic residue can have at least some of the positive binding interactions of the corresponding terminal monosaccharide residue, especially close to the position of 6- hydroxyl/carboxyl/amide of the terminal monosaccharide residue.
  • analogs are produced from amine group by replacement of hydroxyl group at position 3 of Gal/GalNAc ⁇ 4.
  • the present invention is specifically directed to the screening of analogs comprising terminal cyclic molecule on terminal 3-position of Gal/GalNAc in the trisaccharide epitope for binding to Helicobacter pylori.
  • the terminal cyclic molecule is preferably a six membered organic residue, and more preferably it also comprises a carboxylic acid, an amide or alkyl amide structure similar to the stractures terminal and/or in the middle hexuronic acids in the formulas according the invention.
  • Alternatively terminal uronic acid amide or analogue or derivative is 1-3 -linked to Gal, which is linked to the GlcNAc mimicking structure.
  • monosaccharide rings may be replaced by rings such as cyclohexane or cyclopentane, aromatic rings including benzene ring, heterocyclic ring stractures may comprise beside oxygen for example nitrogen and sulphur atoms.
  • the ring stractures may be interconnected by tolerated linker groups.
  • Typical mimetic structure may also comprise peptide analog- structures for the oligosaccharide sequence or part of it.
  • the present invention is also directed to the design and/or screening of peptide analogs for the oligosaccharide sequences. Furthermore the present invention is directed for screening of DNA or RNA-based analogues, for example so called aptamers, of the oligosaccharide sequences according to the invention.
  • the effects of the active groups to binding activity are cumulative and lack of one group could be compensated by adding an active residue on the other side of the molecule.
  • Molecular modelling preferably by a computer, can be used to produce analog stractures for the Helicobacter pylori binding oligosaccharide sequences according to the invention.
  • results from the molecular modelling of several oligosacharide sequences are given in examples and the same or similar methods, besides NMR and X-ray crystallography methods, can be used to obtain stractures for other binding oligosaccharide sequences or analogues or derivatives according to the invention.
  • the analogues or derivatives of the oligosaccharide stractures having same or similar conformations with the oligosaccharide stractures according to the invention are selected from computer assisted molecular modeling results or screening database or databases containing three dimensional stractures of molecules, these methods are referred as computerized screening methods.
  • the present invention is further directed to the testing oligosaccharide stractures and analogues and derivatives thereof which are selected by the computerized screening methods for binding to other pathogenic microbes or virases or toxins having a binding specificity similar to the binding specificity of Helicobacter pylori with regard to one or several oligosaccharide sequences according to the invention.
  • the oligosaccharide structures and analogues and derivatives selected by the computerized screening methods are tested for binding to toxin A of Clostridium difficile.
  • the analogue molecules can be synthetically produced or obtained from natural sources. Molecules can also be produced virtually in computers and part of the screening of the active molecules can also be performed in silico.
  • the present invention is also directed to the searching of Helicobacter pylori-binding and/or inhibiting analogues and/or derivatives for the oligosaccharide stractures according to the invention by computerized fitting of a carbohydrate stracture, analogue or derivative to a carbohydrate binding site on H. pylori.
  • the Helicobacter pylori-binding oligosacchride sequence, analogues or derivatives thereof are "docked" by methods of molecular modeling to the carbohydrate binding molecule(s) of Helicobacter pylori, most probably to lectins of the bacterium and additional binding interactions are searched.
  • the computerized docking of a three dimensional structure of the oligosaccharide sequence on a three dimensional model of a carbohydrate binding site further helps the design of binding active analogues by allowing determination of binding interactions and positions for possible additional binding interactions.
  • the method is also directed to the comparison of the binding of the oligosaccharide stractures and analogues and derivatives thereof by the computerized docking methods.
  • the present invention is further directed to the testing of oligosaccharide structures and analogues and derivatives thereof by the computerized docking methods for binding to other pathogenic microbes or viruses or toxins having a binding specificity similar to the binding specificity of Helicobacter pylori with regard to one or several oligosaccharide sequences according to the invention.
  • the oligosaccharide stractures and analogues and derivatives thereof are tested for binding to toxin A of Clostridium difficile by the computerized docking methods.
  • the monovalent, oligovalent or polyvalent oligosaccharides can be activated to have higher activity towards lectins by making a derivative of the oligosaccharide by combinatorial chemistry.
  • a library is created by substituting one or few residues in the oligosaccharide sequence, it can be considered as a derivative library.
  • the library is created from the analogs of the oligosaccharide sequences described by the invention, it can be considered as an analog library.
  • a combinatorial chemistry library can be built on the oligosaccharide or its precursor or on glycoconjugates according to the invention.
  • oligosaccharides with variable reducing end can be produced by so called carbohydrid technology.
  • the present invention is directed to the design and production of a combinatorial chemistry library, a multide of chemical analogues and/or derivatives of the oligosaccharide stractures according to the invention, and testing these for binding or inhibition of Helicobacter pylori.
  • the present invention is further directed to the testing of the combinatorial chemistry library for binding to other pathogenic microbes or virases or toxins having a binding specificity similar to the binding specificity of Helicobacter pylori with regard to one or several oligosaccharide sequences according to the invention.
  • the combinatorial chemistry library is tested for binding to toxin A of Clostridium difficile.
  • a combinatorial chemistry library is conjugated to the Helicobacter pylori binding substances described by the invention.
  • the library comprises at least 6 different molecules.
  • the combinatorial chemistry modifications are produced by different amides from carboxylic acid group on R 8 or R 9 according to Formula 9.
  • Group to be modified in R 8 may also be an aldehyde or amine or another type of reactive group.
  • Such library is preferred for use of assaying microbial binding to the oligosaccharide sequences according to the invention.
  • Amino acids or collections of organic amides are commercially available, which substances can be used for the synthesis of combinatorial library of uronic acid amides.
  • a high affinity binder could be identified from the combinatorial library for example by using an inhibition assay, in which the library compounds are used to inhibit the bacterial binding to the glycolipids or glycoconjugates described by the invention.
  • Structural analogs and derivatives preferred according to the invention can inhibit the binding of the Helicobacter pylori binding oligosaccharide sequences according to the invention to Helicobacter pylori.
  • the Helicobacter pylori binding sequence is described as an oligosaccharide sequence.
  • the oligosaccharide sequence defined here can be a part of a natural or synthetic glycoconjugate or a free oligosaccharide or a part of a free oligosaccharide.
  • Such oligosaccharide sequences can be bonded to various monosaccharides or oligosaccharides or polysaccharides on polysaccharide chains, for example, the saccharide sequence is expressed as part of a bacterial polysaccharide.
  • numerous natural modifications of monosaccharides are known as exemplified by O-acetyl or sulphated derivatives of the oligosaccharide sequences.
  • the oligosaccharide sequence means terminal non-reducing end oligosaccharide sequence which is not modified by any other monosaccharide residue, except optionally at the reducing end.
  • the term oligosaccharide sequence includes structural analogues and derivatives of the oligosaccharide stractures according to the invention, preferably as described by the invention, having same or similar binding activity with regard to the H. pylori.
  • the Helicobacter pylori binding substance defined here can comprise the oligosaccharide sequence described as a natural or synthetic glycoconjugate or part thereof or a corresponding free oligosaccharide or a part of a free oligosaccharide.
  • the Helicobacter pylori binding substance can also comprise a mixture of the Helicobacter pylori binding oligosaccharide sequences.
  • the Helicobacter pylori binding oligosaccharide sequences can be synthesized enzymatically by glycosyltransferases, or by transglycosylation catalyzed by glycosidase or transglycosidase enzymes (Ernst et al, 2000). Specifities of these enzymes and the use of co-factors can be engineered. Specific modified enzymes can be used to obtain more effective synthesis, for example, glycosynthase is modified to perform transglycosylation only. Organic synthesis of the saccharides and the conjugates described herein or compounds similar to these are known (Ernst et al, 2000).
  • Saccharide materials can be isolated from natural sources and modified chemically or enzymatically into the Helicobacter pylori binding compounds. Natural oligosaccharides can be isolated from milks of various ruminants. Transgenic organisms, such as cows or microbes, expressing glycosylating enzymes can be used for the production of the saccharides.
  • the uronic acid monosaccharide residues described in the invention can be obtained by methods known in the art.
  • the hydroxyl of the 6-carbon of N- acetylglucosamine or N-acetylgalactosamines can be chemically oxidized to carboxylic acid.
  • the oxidation can be performed to a properly protected oligosaccharide or monosaccharide or even to a non-protected carbohydrate.
  • a non-protected polymer or oligomer comprising hexoses, N-acetylhexosamines or hexosamines, wherein the linkage between the monosaccharides is not between carbon 6 atoms, is
  • oligomers or polymers of cellulose, starch or other glucans with 1-2 or 1-3 or 1-4 linkages chitin (GlcNAc polymer) or chitosan (GlcN polymer), which are commercially available in large scale or N- acetylgalactosamine/galactosamine polysaccharides (for example, ones known from a bacterial source) is oxidized to a corresponding 1-4-linked saccharide.
  • This method can also be applied to galactan polymers.
  • Derivatives of uronic acid can be produced also from natural polymers comprising uronic acids such as pectins or glucuronic acid containing bacterial polysaccharides including N-acetylheparin, hyaluronic and chondroitin type animal or bacterial polysaccharides. This method involves
  • Chemical and enzymatic methods are also known to oxidize primary alcohol on carbon 6 of the polysaccharide to aldehyde or to carboxylic acid.
  • An aldehyde can be further derivatized, for example, to amine by reductive amination.
  • Preferably terminal Gal or GalNAc is oxidized by a primary alcohol oxidizing enzyme-like galactose oxidase and can then be further derivatized, for example, by amines.
  • the uronic acid residues can be conjugated to disaccharides or oligosaccharides by standard methods of organic chemistry.
  • GlcA can be linked by a glucuronyl transferase transferring a GlcA from UDP-GlcA to terminal Lac(NAc).
  • Monosaccharide derivatives mimicking N-acetylhexosamines could be produced from a polymer or an oligomer comprising hexosamines or other monosaccharides with free primary amine groups by method involving:
  • Chitosan and oligosaccharides thereof are an example of an amine comprising polymer or oligomer.
  • the method to produce carboxylic acid containing, 6-aldehydo comprising, amine and/or amide comprising monosaccharide/monosaccharides involves following steps 1. optionally introducing an carboxylic acid or 6-aldehydo group to a carbohydrate polymer wherein primary hydroxyl is available for modification 2. derivatization of carboxylic acid groups or 6-aldehydo groups or primary amine groups of the polymer to secondary or tertiary amines or to amides, when step 1 is applied, step 2 is optional. 3. hydrolysis of the polymer to corresponding monosaccharides.
  • the hydrolysis to monosaccharides may also be partial and produce useful disaccharide or oligosaccharide to produce analog substances. Preferably the hydrolysis produces at least 30 % of monosaccharides.
  • Methods to produce the chemical steps are known in the art. For example oxidation of the polysaccharides to corresponding monoaccharides can be performed as described by Muzzarelli et al 1999 and 2002. These methods are preferred to the use of non-protected monosaccharides, because the protection or reactive reducing ends of the monosaccharides is avoided.
  • the oligosaccharide sequences comprising GlcA ⁇ 3Lac or GlcA ⁇ 3LacNAc are effectively synthesised by transglycosylation using a specific glucuronidase such as glucuronidase from bovine liver. It was realized that the enzyme can site-specifically transfer from ⁇ l-3 linkage to Gal ⁇ 4GlcNAc and Gal ⁇ 4Glc with unexpectedly high yields for a transglycosylation reaction. In general such selectivity and yields close 30 % or more are not obtained in transglycosylation reactions.
  • R 8 is preferably carboxylic acid amide, such as -CO-NH 2 , methylamide or ethyalamide, hydroxymethyl (-CH 2 -OH) or a carboxylic acid group or an ester thereof, such as methyl or ethyl ester.
  • the carboxylic acid amide may comprise an alternative linkage to the polyvalent carrier Z comprising an amine such as chitosan or galactosamine polysaccharide or Z comprising a primary amine containing spacer, preferably a hydrophilic spacer.
  • the stracture in R 8 can be also a mimicking structure known in the art to ones described above. For example secondary or tertiary amines or amidated secondary amine can be used.
  • R 9 is preferably hydroxymethyl or carboxylic acid amide, but it can be used for derivatisations as described for R 8 .
  • R 3 is hydroxyl, acetamido or acetamido group mimicking group, such as C ⁇ - 6 alkylamides, arylamido, secondary amine, preferentially N-ethyl or N-methyl, O-acetyl, or O-alkyl for example O-ethyl or O-methyl.
  • R 7 and Rio are same as R 3 but more preferentially acetamido or acetamido mimicking group.
  • R may also comprise preferentially a six-membered ring stracture mimicking Gal ⁇ 4- terminal.
  • the bacterium binding substances are preferably represented in clustered form such as by glycolipids on cell membranes, micelles, liposomes, or on solid phases such as TCL-plates used in the assays.
  • clustered representation with correct spacing creates high affinity binding.
  • Helicobacter pylori binding epitopes or naturally occurring, or a synthetically produced analogue or derivative thereof having a similar or better binding activity with regard to Helicobacter pylori it is also possible to use a substance containing the bacterium binding substance such as a receptor active ganglioside described in the invention or an analogue or derivative thereof having a similar or better binding activity with regard to Helicobacter pylori.
  • the bacterium binding substance may be a glycosidically linked terminal epitope of an oligosaccharide chain.
  • the bacterium binding epitope may be a branch of an oligosaccharide chain, preferably a polylactosamine chain.
  • the Helicobacter pylori binding substance may be conjugated to an antibiotic substance, preferably a penicillin type antibiotic.
  • the Helicobacter pylori binding substance targets the antibiotic to Helicobacter pylori.
  • Such conjugate is beneficial in treatment because a lower amount of antibiotic is needed for treatment or therapy against Helicobacter pylori, which leads to lower side effect of the antibiotic.
  • the antibiotic part of the conjugate is aimed at killing or weaken the bacteria, but the conjugate may also have an antiadhesive effect as described below.
  • the bacterium binding substances can be used to treat a disease or condition caused by the presence of the Helicobacter pylori. This is done by using the Helicobacter pylori binding substances for anti- adhesion, i.e. to inhibit the binding of Helicobacter pylori to the receptor epitopes of the target cells or tissues.
  • the Helicobacter pylori binding substance or pharmaceutical composition When the Helicobacter pylori binding substance or pharmaceutical composition is administered it will compete with receptor glycoconjugates on the target cells for the binding of the bacteria. Some or all of the bacteria will then be bound to the Helicobacter pylori binding substance instead of the receptor on the target cells or tissues.
  • the bacteria bound to the Helicobacter pylori binding substances are then removed from the patient (for example by the fluid flow in the gastrointestinal tract), resulting in reduced effects of the bacteria on the health of the patient.
  • the substance used is a soluble composition comprising the Helicobacter pylori binding substances.
  • the substance can be attached to a carrier substance which is preferably not a protein. When using a carrier molecule several molecules of the Helicobacter pylori binding substance can be attached to one carrier and inhibitory efficiency is improved.
  • the target cells are primarily epithelial cells of the target tissue, especially the gastrointestinal tract, other potential target tissues are for example liver and pancreas. Glycosylation of the target tissue may change because of infection by a pathogen (Karlsson et al, 2000). Target cells may also be malignant, transformed or cancer/tumour cells in the target tissue. Transformed cells and tissues express altered types of glycosylation and may provide receptors to bacteria. Binding of lectins or saccharides (carbohydrate-carbohydrate interaction) to saccharides on glycoprotein or glycolipid receptors can activate cells, in case of cancer/malignant cells this may be lead to growth or metastasis of the cancer.
  • oligosaccharide epitopes described herein such as GlcNAc ⁇ 3Gal ⁇ 4GlcNAc (Hu, J. et al, 1994), Gal ⁇ 3Gal ⁇ 4GlcNAc (Castronovo et al, 1989), and neutral and sialylated polylactosamines from malignant cells (Stroud et al, 1996), have been reported to be cancer-associated or cancer antigens. Oligosaccharide chains containing substances have also been described from lymphocytes (Nivier et al, 1993). Helicobacter pylori is associated with gastric lymphoma.
  • the substances of the invention can be used to prevent binding of Helicobacter pylori to premalignant or malignant cells and activation of cancer development or metastasis. Inhibition of the binding may cure gastric cancer, especially lymphoma.
  • the Helicobacter pylori binding oligosaccharide sequence has been reported in the stracture
  • Glc ⁇ Ac ⁇ 3Gal ⁇ 4Glc ⁇ Ac ⁇ 6Gal ⁇ Ac from human gastric mucins. This mucin epitope and similar O-glycan glycoforms are most probably natural high affinity receptors for Helicobacter pylori in human stomach. This was also indicated by high affinity binding of an analogous sequence GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc as neoglycolipid to Helicobacter pylori and that the sequence
  • GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6Gal has also some binding activity towards Helicobacter pylori in the same assay. Therefore the preferred oligosaccharide sequences includes O-glycans and analogues of O-glycan sequences such as GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc/GalNAc/Gal, GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc/GalNAc/Gal ⁇ Ser/Thr,
  • HOS Helicobacter pylori binding oligosaccharide sequences
  • trisaccharide epitopes are also especially preferred when linked from the reducing end to form stractures OS ⁇ 6Gal(NAc)o- ⁇ or OS ⁇ 6Glc(NAc) 0 _ ⁇ or OS ⁇ 6Gal(NAc) 0 - ⁇ Ser/Thr or OS ⁇ 6Glc(NAc) 0 - ⁇ Ser/Thr.
  • the Ser or Thr- compounds or analogue thereof or the reducing oligosaccharides are also preferred when linked to polyvalent carrier.
  • the reducing oligosaccharides can be reductively linked to the polyvalent carrier.
  • Target cells also includes blood cells, especially leukocytes. It is known that Helicobacter pylori strains associated with peptic ulcer, as the strain mainly used here, stimulates an inflammatory response from granulocytes, even when the bacteria are nonopsonized (Rautelin et al, 1994a,b). The initial event in the phagocytosis of the bacterium most likely involves specific lectin-like interactions resulting in the agglutination of the granulocytes (Ofek and Sharon, 1988). Subsequent to the phagocytotic event oxidative burst reactions occur which may be of consequence for the pathogenesis of Helicobacter pylori-associated diseases (Babior, 1978).
  • glycosphingolipids having repeating N-acetyllactosamine units have been isolated and characterized from granulocytes (Fukuda et ⁇ , 1985; Stroud et ⁇ , 1996) and may thus act as potential receptors for Helicobacter pylori on the white blood cell surface. Furthermore, also the X glycosphingolipid has been isolated from the same source (Teneberg, S., unpublished).
  • the present invention confirms the presence of receptor saccharides on human erythrocytes and granulocytes which can be recognized by an N- acetyllactosamine specific lectin and by a monoclonal antibody (x 2 , GalNAc ⁇ 3Gal ⁇ 4GlcNAc-).
  • the Helicobacter pylori binding substances can be useful to inhibit the binding of leukocytes to Helicobacter pylori and in prevention of the oxidative burst and/or inflammation following the activation of leukocytes.
  • Helicobacter pylori can bind several kinds of oligosaccharide sequences. Some of the binding by specific strains may represent more symbiotic interactions which do not lead to cancer or severe conditions.
  • the present data about binding to cancer-type saccharide epitopes indicates that the Helicobacter pylori binding substance can prevent more pathologic interactions, in doing this it may leave some of the less pathogenic Helicobacter pylori bacteria/strains binding to other receptor stractures. Therefore total removal of the bacteria may not be necessary for the prevention of the diseases related to Helicobacter pylori.
  • the less pathogenic bacteria may even have a probiotic effect in the prevention of more pathogenic strains of Helicobacter pylori.
  • Helicobacter pylori contains large polylactosamine oligosaccharides on its surface which at least in some strains contains non- fucosylated epitopes which can be bound by the bacterium as described by the invention.
  • the substance described herein can also prevent the binding between Helicobacter pylori bacteria and that way inhibit bacteria for example in process of colonization.
  • the Helicobacter pylori binding substance optionally with a carrier, in a pharmaceutical composition, which is suitable for the treatment of a condition due to the presence of Helicobacter pylori in a patient or to use the Helicobacter pylori binding substance in a method for treatment of such conditions.
  • conditions treatable according to the invention are chronic superficial gastritis, gastric ulcer, duodenal ulcer, non-Hodgkin lymphoma in human stomach, gastric adenocarcinoma, and certain pancreatic, skin, liver, or heart diseases, sudden infant death syndrome, autoimmune diseases including autoimmune gastritis and pernicious anaemia and non-steroid anti- inflammatory drag (NSAID) related gastric disease, all, at least partially, caused by the Helicobacter pylori infection.
  • NSAID non-steroid anti- inflammatory drag
  • the pharmaceutical composition containing the Helicobacter pylori binding substance may also comprise other substances, such as an inert vehicle, or pharmaceutically acceptable carriers, preservatives etc, which are well known to persons skilled in the art.
  • the Helicobacter pylori binding substance can be administered together with other drags such as antibiotics used against Helicobacter pylori.
  • the Helicobacter pylori binding substance or pharmaceutical composition containing such substance may be administered in any suitable way, although an oral administration is preferred.
  • treatment used herein relates both to treatment in order to cure or alleviate a disease or a condition, and to treatment in order to prevent the development of a disease or a condition.
  • the treatment may be either performed in a acute or in a chronic way.
  • patient or "subject”, as used herein, relates to any human or non-human mammal in need of treatment according to the invention.
  • the Helicobacter pylori binding substance it is also possible to use the Helicobacter pylori binding substance to identify one or more adhesins by screening for proteins or carbohydrates (by carbohydrate- carbohydrate interactions) that bind to the Helicobacter pylori binding substance.
  • the carbohydrate binding protein may be a lectin or a carbohydrate binding enzyme. The screening can be done for example by affinity chromatography or affinity cross linking methods (liver et al, 1998).
  • the binding substance should be suitable for such use such as a humanized antibody or a recombinant glycosidase of human origin which is non-immunogenic and capable of cleaving the terminal monosaccharide residue/residues from the Helicobacter pylori binding substances.
  • lectins such as Erythrina cristagalli and Erythrina corallodendron (Teneberg et al, 1994.
  • the binding substance should be suitable for such use such as a humanized antibody or a recombinant glycosidase of human origin which is non-immunogenic and capable of cleaving the terminal monosaccharide residue/residues from the Helicobacter pylori binding substances.
  • many naturally occuring lectins and glycosidases originating for example from food are tolerated.
  • the Helicobacter pylori binding substance is possible to use as part of a nutritional composition including food- and feedstuff. It is preferred to use the Helicobacter pylori binding substance as a part of so called functional or functionalized food.
  • the said functional food has a positive effect on the person's or animal's health by inhibiting or preventing the binding of Helicobacter pylori to target cells or tissues.
  • the Helicobacter pylori binding substance can be a part of a defined food or functional food composition.
  • the functional food can contain other acceptable food ingredients accepted by authorities such as Food and Drag Administration in the USA.
  • the Helicobacter pylori binding substance can also be used as a nutritional additive, preferably as a food or a beverage additive to produce a functional food or a functional beverage.
  • the food or food additive can also be produced by having ,e.g., a domestic animal such as a cow or other animal produce the Helicobacter pylori binding substance in larger amounts naturally in its milk. This can be accomplished by having the animal overexpress suitable glycosyltransferases in its milk. A specific strain or species of a domestic animal can be chosen and bred for larger production of the Helicobacter pylori binding substance.
  • the Helicobacter pylori binding substance for a nutritional composition or nutritional additive can also be produced by a micro-organisms such as a bacteria or a yeast.
  • the Helicobacter pylori binding substance is especially useful to have the Helicobacter pylori binding substance as part of a food for an infant, preferably as a part of an infant formula.
  • Many infants are fed by special formulas in replacement of natural human milk.
  • the formulas may lack the special lactose based oligosaccharides of human milk, especially the elongated ones such as lacto-N-neotetraose, Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4Glc, and its derivatives.
  • the natural type oligosaccharide derived from desulphated chondroitin, and hyaluronic acid are preferred for infant formulas, other functional foods and food additive.
  • carboxylic acid reduced hyaluronic acid and chondroitin oligosaccharides according to the invention are considered useful for use in infant formulas as close analogs of the natural type polylactosamines and lactose based milk oligosaccharides, the chondroitin saccharides has also homology with core 1 stractures of O-linked gly cans, moreover disaccharide GalNAc ⁇ 4Glc is a component of bovine milk.
  • oligosaccharides are preferred for infant foods, other functional foods, feeds and nutritional additives: GalNAc ⁇ 4Glc, Glc ⁇ 3GalNAc ⁇ 4Glc, Glc ⁇ 3GalNAc ⁇ 4Glc ⁇ 3GalNAc ⁇ 4Glc, Glc ⁇ 3GalNAc ⁇ 4Glc ⁇ 3GalNAc ⁇ 4Glc ⁇ 3GalNAc ⁇ 4Glc, Glc ⁇ 3GalNAc ⁇ 4Glc ⁇ 3GalNAc, Glc ⁇ 3GalNAc ⁇ 4Glc ⁇ 3GalNAc ⁇ 4Glc ⁇ 3GalNAc,
  • Helicobacter pylori is especially infective with regard to infants or young children, and considering the diseases it may later cause it is reasonable to prevent the infection. Helicobacter pylori is also known to cause sudden infant death syndrome, but the strong antiobiotic treatments used to eradicate the bacterium may be especially unsuitable for young children or infants.
  • the Helicobacter pylori binding substance in the diagnosis of a condition caused by an Helicobacter pylori infection. Diagnostic uses also include the use of the Helicobacter pylori binding substance for typing of Helicobacter pylori.
  • the substance may be included in, e.g., a probe or a test stick, optionally constituting a part of a test kit. When this probe or test stick is brought into contact with a sample containing Helicobacter pylori, the bacteria will bind the probe or test stick and can be thus removed from the sample and further analyzed.
  • the non-reducing end terminal monosaccharide residue in the preferred trisaccharide sequences of the invention can contain a carboxylic acid group on the carbon 6 (terminal monosaccahride residue is a uronic acid, HexA or HexANAc, wherein Hex is Gal or Glc) or a derivative of the carbon 6 of the HexA(NAc) residue or a derivative of the carbon 6 of the corresponding Hex(NAc) residue.
  • Such terminal residues includes preferably ⁇ 3 -linked glucuronic acid and more preferably 6-amides such as methylamide thereof. Therefore analogs and derivatives of the sequence can be produced by changing or derivatising the terminal 6-position of the trisaccharide epitopes.
  • Preferred Helicobacter pylori binding substances are preferably ⁇ 3 -linked glucuronic acid and more preferably 6-amides such as methylamide thereof.
  • the oligosaccharide sequences according to the invention were found to be unexpectedly effective binders when presented on thin layer surface. This method allows polyvalent presentation of the glycolipid sequences. The surprisingly high activity of the polyvalent presentation of the oligosaccharide sequences makes polyvalency a preferred way to represent the oligosaccharide sequences of the invention.
  • glycolipid stractures are naturally presented in a polyvalent form on cellular membranes. This type of representation can be mimicked by the solid phase assay described below or by making liposomes of glycolipids or neoglycolipids.
  • the present novel neoglycolipids produced by reductive amination of hydrophobic hexadecylaniline were able to provide effective presentation of the oligosaccharides.
  • Most previously known neoglycolipid conjugates used for binding of bacteria have contained a negatively charged groups, such as phosphor ester of phosphadityl ethanolamine neoglycolipids. Problems of such compounds are negative charge of the substance and natural biological binding involving the phospholipid structure. Negatively charged molecules are known to be involved in numerous non-specific bindings with proteins and other biological substances. Moreover, many of these stractures are labile and can be enzymatically or chemically degraded.
  • the present invention is directed to the non-acidic conjugates of oligosaccharide sequences meaning that the oligosaccharide sequences are linked to non-acidic chemical structures.
  • the non-acidic conjugates are neutral meaning that the oligosaccharide sequences are linked to neutral, non-charged, chemical structures.
  • the preferred conjugates according to the invention are polyvalent substances.
  • bioactive oligosaccharide sequences are often linked to carrier structures by reducing a part of the receptor active oligosaccharide stracture.
  • Hydrophobic spacers containing alkyl chains (-CH 2 -) n and/or benzyl rings have been used.
  • hydrophobic stractures are in general known to be involved in nonspecific interactions with proteins and other bioactive molecules.
  • neoglycolipid data of the examples below show that under the experimental conditions used in the assay the hexadecylaniline parts of the neoglycolipid compounds do not cause non-specific binding for the studied bacterium.
  • the hexadecylaniline part of the conjugate forms probably a lipid layer like stracture and is not available for the binding.
  • the invention shows that reducing a monosaccharide residue belonging to the binding epitope may destroy the binding. It was further realized that a reduced monosaccharide can be used as a hydrophilic spacer to link a receptor epitope and a polyvalent presentation structure.
  • the bioactive oligosaccharide via a hydrophilic spacer to a polyvalent or multivalent carrier molecule to form a polyvalent or oligovalent/multivalent stracture.
  • All polyvalent (comprising more than 10 oligosaccharide residues) and oligovalent/multivalent structures (comprising 2-10 oligosaccharide residues) are referred here as polyvalent structures, though depending on the application oligovalent/multivalent constructs can be more preferred than larger polyvalent stractures.
  • the hydrophilic spacer group comprises preferably at least one hydroxyl group. More preferably the spacer comprises at least two hydroxyl groups and most preferably the spacer comprises at least three hydroxyl groups.
  • the hydrophilic spacer group is preferably a flexible chain comprising one or several -CHOH- groups and/or an amide side chain such as an acetamido -NHCOCH or an alkylamido.
  • the hydroxyl groups and/or the acetamido group also protects the spacer from enzymatic hydrolysis in vivo.
  • the term flexible means that the spacer comprises flexible bonds and do not form a ring structure without flexibility.
  • a reduced monosaccharide residues such as ones formed by reductive amination in the present invention are examples of flexible hydrophilic spacers.
  • the flexible hydrophilic spacer is optimal for avoiding nonspecific binding of neoglycolipid or polyvalent conjugates. This is essential optimal activity in bioassays and for bioactivity of pharmaceuticals or functional foods, for example.
  • a general formula for a conjugate with a flexible hydrophilic linker has the following Formula 10:
  • Li and L are linking groups comprising independently oxygen, nitrogen, sulphur or carbon linkage atom or two linking atoms of the group forming linkages such as -O-, -S-, -CH 2 -, -N-, -N(COCH3)-, amide groups -CO-NH- or-NH-CO- or -N-N- (hydrazine derivative) or amino oxy-linkages -O-N- and -N-O-.
  • pl, p2, p3, and p4 are independently integers from 0-7, with the proviso that at least one of pl, p2, p3, and p4 is at least 1.
  • CH ⁇ _ 2 OH in the branching term ⁇ CH ⁇ . 2 OH ⁇ p ⁇ means that the chain terminating group is CH 2 OH and when the pl is more than 1 there is secondary alcohol groups -CHOH- linking the terminating group to the rest of the spacer.
  • R is preferably acetyl group (-COCH 3 ) or R is an alternative linkage to Z and then L 2 is one or two atom chain terminating group, in another embodiment R is an analog forming group comprising C ⁇ - 4 acyl group (preferably hydrophilic such as hydroxy alkyl) comprising amido stracture or H or C ⁇ - 4 alkyl forming an amine. And m > 1 and Z is polyvalent carrier. OS and X are defined in Formula 9.
  • pi is 0.
  • the invention shows that open chain forms of a monosaccharide are effective H. pylori binding stractures at the reducing end. These stractures can mimic ceramides with hydroxylfattyacid.
  • the hydrophilic linker according to Formula 10 comprise a hydroxyl group at the same distance from the reducing end oligosaccharide sequence as the corresponding hydroxyl group is on ceramide from the reducing end oligosaccharide in natural glycosphingolipids with hydroxylfattyacid.
  • Preferred polyvalent stractures comprising a flexible hydrophilic spacer according to formula 2 include Helicobacter pylori binding oligosaccharide sequence (OS) ⁇ l-3 linked to Gal ⁇ 4Glc(red)-Z, and OS ⁇ 6GlcNAc(red)-Z and OS ⁇ 6GalNAc(red)-Z., where "(red)” means the amine linkage stracture formed by reductive amination from the reducing end monosaccharides and an amine group of the polyvalent carrier Z.
  • OS Helicobacter pylori binding oligosaccharide sequence
  • the oligosaccharide group is preferably linked in a polyvalent or an oligovalent form to a carrier which is not a protein or peptide to avoid antigenicity and possible allergic reactions, preferably the backbone is a natural non-antigenic polysaccharide.
  • the novelty of the glucuronic acid derivative containing oligosaccharide sequences makes them especially preferred.
  • the natural type of the sequences described by the invention can be cleaved by glycosidase enzymes which reduces usefulness of these especially when used in human and animal body.
  • Glycosidase enzymes cleaving the sequences are known to be active in human gastrointestinal tract.
  • glycosidases such as N-acetylhexosaminidases or galactosidases has been described as digestive enzyme and are also present in food stuffs. Ex vivo uses of the present invention
  • the present invention can be used for inhibition of pathogens especially Helicobacter pylori ex vivo and such method have use in disinfection and preservevation type applications.
  • Glycolipid and carbohydrate nomenclature is according to recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (Carbohydrate Res. 1998, 312, 167; Carbohydrate Res. 1997, 297, 1; Eur. J. Biochem. 1998, 257, 29).
  • Gal, Glc, GlcNAc, and Neu5Ac are of the D-configuration, Fuc of the L-configuration, and all the monosaccharide units in the pyranose form.
  • Glucosamine is referred as GlcN or GlcNH 2 and galactosamine as GalN or GalNH 2 .
  • Glycosidic linkages are shown partly in shorter and partly in longer nomenclature, the linkages of the Neu5 Ac-residues ⁇ 3 and ⁇ 6 mean the same as ⁇ 2-3 and ⁇ 2-6, respectively, and with other monosaccharide residues ⁇ l-3, ⁇ l-3, ⁇ l-4, and ⁇ 1 -6 can be shortened as ⁇ 3, ⁇ 3, ⁇ 4, and ⁇ 6, respectively.
  • Lactosamine refers to N- acetyllactosamine, Gal ⁇ 4GlcNAc
  • sialic acid is N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc) or any other natural sialic acid.
  • Term glycan means here broadly oligosaccharide or polysaccharide chains present in human or animal glycoconjugates, especially on glycolipids or glycoproteins.
  • the number before the colon refers to the carbon chain lenght and the number after the colon gives the total number of double bonds in the hydrocarbon chain.
  • Abbreviation GSL refers to glycosphingolipid.
  • Abbreviations or short names or symbols of glycosphingolipids are given in the text and in Tables 1 and 2.
  • Helicobacter pylori refers also to the bacteria similar to Helicobacter pylori.
  • GlcA is glucuronic acid and derivatives of carbon 6 of glucose or glucuronic acid
  • Gal A is galacturonic acid and derivatives of carbon 6 of galactose or galacturonic acid
  • GlcANAc is N-acetylglucuronic acid and derivatives of carbon 6 of N-acetylghicosamine or is N-acetylglucosamine uronic acid
  • GalANAc is N-acetylgalactosamine uronic acid and derivatives of carbon 6 of N- acetylgalactosamine or N-acetylgalactosamine uronic acid.
  • terminal oligosaccharide sequence indicates that the oligosaccharide is not substituted to the non-reducing end terminal residue by another monosaccharide residue.
  • ⁇ 3/ ⁇ 3 indicates that the adjacent residues in an oligosaccharide sequence can be either ⁇ 3- or ⁇ 3- linked to each other.
  • Ham's F12 medium from Gibco (U.K.), ⁇ S-methionine from Amersham (U.K.) and FCS (fetal calf serum) was from Sera-Lab (England).
  • the clinical isolates of Helicobacter pylori strains 002 and 032 obtained from patients with gastritis and duodenal ulcer, respectively, were a generous gift from Dr. D. Danielsson, Orebro Medical Center, Sweden.
  • Type strain 17875 was from Culture Collection, University of Goteborg (CCUG).
  • Glycosphingolipids The pure glycosphingolipids of the experiment shown in Figs. 7A and 7B were prepared from total acid or non-acid fractions from the sources listed in Table 1 as described in (Karlsson, 1987). In general, individual glycosphingolipids were obtained by acetylation (Handa, 1963) of the total glycosphingolipid fractions and separated by repeated silicic acid column chromatography, and subsequently characterized structurally by mass spectrometry (Samuelsson et al., 1990), NMR (Falk et al, 1979a,b,c; Koerner Jr et al, 1983) and degradative procedures (Yang and Hakomori, 1971; Stellner et al, 1973). Glycolipids derived from rabbit thymys are described below.
  • the Helicobacter pylori strains were stored at -80 °C in tryptic soy broth containing 15% glycerol (by volume). The bacteria were initially cultured on GAB-CAMP agar (Soltesz et al, 1988) under humid (98%) microaerophilic conditions (O 2 : 5-7%, CO 2 : 8-10% and N 2 : 83-87%) at 37 °C for 48-72 h.
  • colonies were inoculated (lxlO 5 CFU/ml) in Ham's F12 (Gibco BRL, U.K.), supplemented with 10% heat-inactivated fetal calf serum (Sera-Lab).
  • 50 ⁇ Ci 35 S-methionine per 10 ml medium was added, and incubated with shaking under microaerophilic conditions for 24 h.
  • Bacterial cells were harvested by centrifugation, and purity of the cultures and a low content of coccoid forms was ensured by phase-contrast microscopy. After two washes with PBS, the cells were resuspended to lxl 0 ⁇ CFU/ml in PBS.
  • TLC bacterial overlay assay Thin-layer chromatography was performed on glass- or aluminum-backed silica gel 60 HPTLC plates (Merck, Darmstadt, Germany) using chloroform/methanol/water 60:35:8 (by volume) as solvent system. Chemical detection was accomplished by anisaldehyde staining (Waldi, 1962). The bacterial overlay assay was performed as described previously (Hansson et al, 1985).
  • Glycosphingolipids (1-4 ⁇ g/lane, or as indicated in the figure legend) were chromatographed on aluminum-backed silica gel plates and thereafter treated with 0.3-0.5% polyisobutylmethacrylate in diethylether/r ⁇ -hexane 1 :3 (by volume) for 1 min, dried and subsequently soaked in PBS containing 2% bovine serum albumin and 0.1% Tween 20 for 2 h. A suspension of radio-labeled bacteria (diluted in PBS to lxl 0 8 CFU/ml and l-5xl0 6 cpm/ml) was sprinkled over the cliromatograms and incubated for 2 h followed by repeated rinsings with PBS. After drying the cliromatograms were exposed to XAR-5 X-ray films (Eastman Kodak Co., Rochester, NY, USA) for 12-72 h.
  • oligosaccharides Synthesis of oligosaccharides.
  • the oligosaccharide GlcNAc ⁇ 3Gal ⁇ 4GlcNAc was synthesised from Gal ⁇ 4GlcNAc (Sigma, St. Louis, USA), GalNAc ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4GlcNAc was synthesized from the trisaccharide by transferring GalNAc from vast excess of UDP-GalNAc (Sigma) by large amounts of ⁇ 4Galactosyltransferase (bovine milk, Calbiochem., CA, USA) and
  • GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6Gal ⁇ 4Glc
  • GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 3Man were synthesised from Gal ⁇ 4GlcNAc ⁇ 6GlcNAc, Gal ⁇ 4GlcNAc ⁇ 6Gal ⁇ 4Glc, Gal ⁇ 4GlcNAc ⁇ 6GlcNAc, and Gal ⁇ 4GlcNAc ⁇ 3Man, respectively, by incubating the acceptor saccharide with human serum ⁇ 3-N-acetylglucosaminyltransf erase and UDP-GlcNAc in presence of 8 mM MnCl 2 and 0.2 mg/ml ATP at 37 degree of Celsius for 5 days in 50 mM TRIS-HCl pH 7.5.
  • Gal ⁇ 4GlcNAc ⁇ 6GlcNAc, Gal ⁇ 4GlcNAc ⁇ 6Gal ⁇ 4Glc, Gal ⁇ 4GlcNAc ⁇ 6GlcNAc, and Gal ⁇ 4GlcNAc ⁇ 3Man were obtained from GlcNAc ⁇ 6GlcNAc (Sigma, St Louis, USA), GlcNAc ⁇ 6Gal ⁇ 4Glc (Sigma), GlcNAc ⁇ 6GlcNAc (HC1 vapor catalyzed acid reversion rection for solid GlcNAc (Sigma) in dessicator and chromatographic purification, NMR, and mass spectrometric analysis), and GlcNAc ⁇ 3Man (DextraLabs Reding, UK) by incubating the acceptor saccharide with ⁇ 4Galactosyltransferase (bovine milk, Calbiochem., CA, USA) and UDP-Gal in presence of 20 mM MnCl 2 for several hours in 50 mM MO
  • GlcNAc ⁇ 3GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4Glc, GlcA ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc, GlcA ⁇ 3Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4Glc were produced from Gal ⁇ 4GlcNAc ⁇ 6GlcNAc, Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4Glc by transglycosylation catalysed by glucuronidase enzyme (bovine testes, Sigma) using GlcA-paranitrophenyl as donor substrate.
  • Glc(A- methylamide) ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc was obtained by methylamidation of the corresponding glucuronyloligosaccharide as described.
  • the oligosaccharides were purified chromatographically and their purity was assessed by MALDI-TOF mass spectrometry and NMR.
  • Gal ⁇ 3Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4Glc was from Dextra laboratories, Reading, UK.
  • the glycolipid GlcA ⁇ 3Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4Glc ⁇ Cer (Wako Pure Chemicals, Osaka, Japan) was reduced to
  • Glc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4Glc ⁇ Cer as described in Lanne et al 1995.
  • the glycolipid derivative Glc(A-methylamide) ⁇ 3Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4Glc ⁇ Cer was produced by amidatation of the carboxylic acid group of the glucuronic acid of GlcA ⁇ 3Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4Glc ⁇ Cer as described in Lanne et al 1995.
  • Beta3-glucuronidation and amidation of Gal ⁇ 4GlcNAc ⁇ 6GlcNAc was ⁇ 3-glucuronidated by transglycosylation using paranitrophenylglucuronide as the donor and bovine ⁇ l,3-glucuronidase as the enzyme.
  • the product was purified by solid-phase extraction using a C-18 material, anion-exchange chromatography and gel filtration.
  • the glucuronic acid unit in GlcA ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc was converted to glucuronmethylamide as follows:
  • the tetrasaccharide was dissolved in pyridine containing 10% water, and 5-fold molar excess of HBTU (Benzotriazole-1-yl- 1,1,3,3,-tetramethyluroniumhexafluorophosphate) and DIPEA (N,N,- dusopropylethylamine) were added. 50-fold molar excess of methylamine was then added and the reaction was allowed to proceed for 48 h at room temperature.
  • HBTU Benzotriazole-1-yl- 1,1,3,3,-tetramethyluroniumhexafluorophosphate
  • DIPEA N,N,- dusopropylethylamine
  • the methylamidated tetrasaccharide was puridied by gel filtration, anion-exchange chromatography as well as hydrophilic interaction chromatoraphy.
  • MALDI-TOF mass spectrum of the purified product shows the expected signals: m/z 798.2 ([M+Naf) and 814.2 ([M+K] + ).
  • the stracture of the molecule was confirmed NMR- spectormetry.
  • the glucuronic acid unit in GlcA ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc was also converted to glucuronamide. Essentially similar method was used as above, except that ammonia was used as the base. The stracture of the molecule was confirmed by MALDI-TOF mass spectrometry and NMR-spectormetry-
  • Chondroitin sulphate A (Sigma) was converted to pyridinium salt by running the sample through cation exchange resin (hydrogen form), after which the solution was titrated to slightly basic pH with pyridine, and then dried in a vacuum sentrifuge. Desulphation was carried out by dissolving the dry sample to DMSO containing
  • Oligosaccharides were produced from the desulphated material by acid hydrolysis in 0.5 M TFA at 60° C for 18 h. In these conditions tetra- and hexasaccharides with GalNAc at their reducing end are effectively produced. Pure tetra- and hexasaccharides were isolated by use of gel filtration and anion-exchange chromatography. NMR-spectorometry reavealed oligosaccharide with non-reducing terminal GlcA and reducing termial GalNAc. The hydrolysis method revealed to be surprisingly selective in cleaving GalNAc ⁇ 4GlcA-bond. The use of volatile acid allows use of oligosaccharide mixrares even without desalting, which is another benefit of using trifluoroacetic acid or similar strong carboxylic acids.
  • Amide groups were introduced to glucuronic acid residues of chondroitin tetrasaccharides and hexasaccharides by using HBTU activator and ammonia as described above. Pure amidated tetra- and hexasaccharides were isolated by use of gel filtration and anion-exchange chromatography. The MALDI-TOF mass spectra of the purified products (Figs. 3 and 4) show the expected signals.
  • amide groups can be introduced to the desulphated chondroitin sulphate polymer, using the methodology essentially as described above.
  • amidation reagents are effectively removed by extensive dialysis, and modified oligosaccharides are produced by acid hydrolysis, as described above.
  • the methods to degradate polysaccharide and amidate glucuronic acid residues described above are also used be (without desulphation step) for commercial hyaluronic acid (for example Sigma) or oligosaccharides such as GlcA ⁇ 3GlcNAc ⁇ 4GlcA ⁇ 3GlcNAc and GlcA ⁇ 3GlcNAc ⁇ 4GlcA ⁇ 3GlcNAc ⁇ 4GlcA ⁇ 3GlcNAc derived from hyaluronic acid (as above).
  • hyaluronidase bovine, Sigma
  • chondroitin or chondroitin sulphate or hyaluronic acid oligosaccharides can be used for production of chondroitin or chondroitin sulphate or hyaluronic acid oligosaccharides.
  • GalNAc ⁇ 3-B5 (no. 25, Table 1), were thus all found to be non-binding. It may be further noticed that the acetamido group of the internal GlcNAc ⁇ 3 in B5 is essential for binding since de-N-acylation of this moiety by treatment with anhydrous hydrazine leads to complete loss of binding (lane 3) as is the case also when neolactotetraosylceramide is similarly treated (no. 6, Table 1).
  • Gal ⁇ 3Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4Glc ⁇ Cer known from human erythrocytes, would be expected to bind the bacterial adhesin and also three other terminal monosaccharides in Helicobacter pylori binding epitopes are trisaccharide binding epitopes, namely GlcNAc ⁇ 3Gal ⁇ 4GlcNAc, Glc ⁇ 3Gal ⁇ 4GlcNAc and
  • Glc ⁇ 3Gal ⁇ 4GlcNAc Such compounds are not known from human tissues so far, but could rather represent analogues of the natural receptor.
  • sequences with changes in the terminal monosaccharide position 6, (for example carboxylic acid, desulfo HNK-1, or amide derivative) and N-asetyl group on position 2 of Gal ⁇ 4 were included to the family of Neolacto type receptors for H. pylori.
  • GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc Gal ⁇ 3Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4Glc, Glc(A- methylamide) ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc, Gal ⁇ 4GlcNAc ⁇ 6GlcNAc, GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6Gal, GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6Gal ⁇ 4Glc
  • GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc
  • GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 3Gal ⁇ 4Glc GlcA ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc, GlcA ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6Glc
  • A-NH 2 indicates the glucuronamides -CO-NH 2 and (A- methylamide) methylamide of the 6-position of glucuronic acid, -CO-NH-CH 3 , were reductively aminated with 4-hexadecylaniline (abbreviation HDA, from Aldrich, Sweden) and/or with another lipid anchor NH 2 -CH 2 -CH 2 -CH 2 -amide- lysineamidated to two palmitates (custom product, Rapp polymere, Germany) by cyanoborohydride (Halina Miller-Podraza, to be published later).
  • HDA 4-hexadecylaniline
  • the products were characterized by mass spectrometry and were confirmed to be conjugates reductively aminated at the reducing end of the molecules to the lipid anchor/anchors.
  • GlcNAc ⁇ 3Gal ⁇ 4GlcNAc ⁇ 6GlcNAc and maltoheptaose neoglycolipids were weakly binding or inactive.
  • the example shows that the terminal tri-or disaccharide epitopes are preferably represented ⁇ 6-HexNAc(Gal, GlcNAc) linked as structures binding to Helicobacter pylori.
  • the reducing end Glc-residue is probably not needed for the binding because the reduction destroys the pyranose ring stracture of the Glc- residue.
  • the intact ring stracture of reducing end GlcNAc is needed for good binding of the trisacharide GlcNAc ⁇ 3Gal ⁇ 4GlcNAc.
  • Example 5 Production of partially sulphated chondroitin oligosaccharides, hyaluronic acid oligosaccharides, polyvalent oligosaccharide and carboxylic acid reduction of an oligosaccharide.
  • Chondroitin sulphate A 500 mg was dissolved in 50 ml of sodium phosphate buffer, pH 6.0, containing 150 mM NaCl. 10 kU hyaluronidase (bovine testes) was added, and the reaction was allowed to proceed at 37 °C. The reaction was monitored by running aliquots in gel filtration chromatography. A fraction containing primarily monosulphated chondroitin octa-, deca- and dodecamers was isolated.
  • the oligosaccharide-dendrimer conjugate was purified by gel filtration chromatography, and the purified product was subjected to MALDI-TOF mass spectrometry (Fig. 6). As the dendrimer has MW of 1687 Da and the increment in size per tetrasaccharide attached to the dendrimer is 776 Da, the product was found to contain on average 7.7 tetrasaccharide units per dendrimer.
  • oligosaccharide mixture was isolated from a hyaluronic acid hydrolysis reaction by gel filtration chromatography. This fraction contained hexa-, octa-, deca- and dodekasaccharides. A sample of this mixture containing 4 ⁇ mol of carboxyl groups was methylesterified in a solution containing 1.6 mmol of CH 3 I (102 ⁇ l), 4 ⁇ mol of DIPEA, 31 ⁇ l H 2 O and 173 ⁇ l DMSO. The reaction was allowed to proceed for 48 h at RT in the dark. The methylesterified oligosaccharide mixture was purified by extensive dialysis.
  • the methylesterified sample was dissolved in 100 ⁇ l of 50 mM ammonium bicarbonate and 3.2 mg of sodium borohydride was added. The reaction was allowed to proceed for 2 h at RT, and the product was isolated by gel filtration chromatography.
  • the MALDI-TOF mass spectrum of the reduced sample is shown in Fig. 7.
  • the major signals in the spectran are as follows: M/z Ion type Suggested composition
  • GlcA ⁇ 3GalNAc ⁇ 4 [GlcA ⁇ 3GalNAc ⁇ 4] 2 .
  • GlcA ⁇ 3GalNAc mixture of octa-, deca-, and dodeca- saccharides
  • TLC plate with acidic oligosaccharides Starburst (PAMAM)-dendrimer was used as carrier as described in WO03002128 and were observed to be binding active with regard to Helicobacter pylori.
  • PAMAM acidic oligosaccharides Starburst
  • glycosphingolipid shorthand nomenclature follows recent recommendations (Nomenclature of glycoproteins, 1988).
  • b The following abbreviations are used for the glycosphingolipid sources: RT, rabbit thymus; HE, human erythrocytes; RE, rabbit erythrocytes; HM, human meconium; RCC, rat colon carcinoma; BB, bovine buttermilk; DSI, dog small intestine, c
  • binding strength is as follows: + denotes a significant darkening of the autoradiogram with 4 ⁇ g applied on the TLC plate, (+) indicates a weak to intermediate darkening while a minus sign signifies no binding.
  • d Prepared from No. 27 by mild acid hydrolysis and No. 10 by subsequent treatment with ⁇ -galactosidase.
  • e Glycosphingolipid Nos. 3, 6, 8 and 14 were prepared from Nos.2, 5, 7 and 13, respectively, by treatment with anhydrous hydrazine.
  • f Prepared from no. 19 by neuramimdase treatment.
  • g Prepared by mild acid hydrolysis of GM1 ganglioside from human brain.
  • h Prepared from No. 22 by incubation in 0.05 M HCl at 80°C for 2 h.
  • Gastroenterology 111, 433-438 Castronovo, N., Colin, C, Parent, B., Foidart, J.-M., Lambotte, R., and Mahieu, P. (1989) J. Natl Cancer In t, 81, 212-216 Clausen, H., Levery, S.B., Kannagi, R. and Hakomori, S.-i. (1986) J. Biol Chem., 261, 1380-1387. Chmiela, M., Wadstrom, T., Folkesson, H., Planeta Malecka, I., Czkwianianc, E.,

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Abstract

L'invention concerne des épitopes de liaison à Helicobacter pylori, à savoir des substances comprenant des séquences oligosaccharidiques Hex2(NAc)β4Glc(A)(NAc) et Hex1(A)(NAc)α/β3Hex2(NAc) (où Hex1 = Gal, Glc ou Man, Hex2 = Gal ou Glc, les éléments entre parenthèses étant facultatifs, et GlcA représente un dérivé d'acide glucuronique). L'invention concerne également l'utilisation desdits épitopes dans des compositions pharmaceutiques et nutritionnelles pour traiter des états pathologiques dus à la présence de Helicobacter pylori chez un sujet, tels que les gastrites, les ulcères gastriques, l'adénocarcinome gastrique, les maladies du foie, du pancréas, de la peau, du coeur, les maladies auto-immunes et la mort subite du nourrisson. La présente invention concerne également l'utilisation du récepteur pour le diagnostic de Helicobacter pylori, un procédé pour produire des oligosaccharides de chondroïtine à partir de sulfates de chondroïtine, ainsi qu'un procédé pour produire de l'acide glucuronique amidé, contenant des oligosaccharides et des monosaccharides, à partir d'acide glucuronique contenant des polysaccharides.
PCT/FI2004/000027 2003-01-20 2004-01-20 Nouveaux epitopes de liaison a helicobacter pylori et utilisation desdits epitopes WO2004065400A1 (fr)

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CN102676613A (zh) * 2012-05-22 2012-09-19 江南大学 一种硫酸软骨素二糖、四糖、六糖的制备方法
CN105879052A (zh) * 2016-06-06 2016-08-24 北京林业大学 一种果胶-多臂聚乙二醇自组装制备纳米药物的方法
US10662212B2 (en) 2014-03-13 2020-05-26 Universitat Basel Carbohydrate ligands that bind to IGM antibodies against myelin-associated glycoprotein
CN113150086A (zh) * 2021-04-22 2021-07-23 成都亿妙生物科技有限公司 幽门螺杆菌HefC重组蛋白及其应用
US11091591B2 (en) 2015-09-16 2021-08-17 Universität Basel Carbohydrate ligands that bind to antibodies against glycoepitopes of glycosphingolipids

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102676613A (zh) * 2012-05-22 2012-09-19 江南大学 一种硫酸软骨素二糖、四糖、六糖的制备方法
CN102676613B (zh) * 2012-05-22 2013-08-07 江南大学 一种硫酸软骨素二糖、四糖、六糖的制备方法
US10662212B2 (en) 2014-03-13 2020-05-26 Universitat Basel Carbohydrate ligands that bind to IGM antibodies against myelin-associated glycoprotein
US11220523B2 (en) 2014-03-13 2022-01-11 Universität Basel Carbohydrate ligands that bind to IgM antibodies against myelin-associated glycoprotein
US11091591B2 (en) 2015-09-16 2021-08-17 Universität Basel Carbohydrate ligands that bind to antibodies against glycoepitopes of glycosphingolipids
CN105879052A (zh) * 2016-06-06 2016-08-24 北京林业大学 一种果胶-多臂聚乙二醇自组装制备纳米药物的方法
CN105879052B (zh) * 2016-06-06 2019-02-15 北京林业大学 一种果胶-多臂聚乙二醇自组装制备纳米药物的方法
CN113150086A (zh) * 2021-04-22 2021-07-23 成都亿妙生物科技有限公司 幽门螺杆菌HefC重组蛋白及其应用
CN113150086B (zh) * 2021-04-22 2022-10-11 成都欧林生物科技股份有限公司 幽门螺杆菌HefC重组蛋白及其应用

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