EP0588852A1 - Derives d'oligosaccharide immunosuppresseurs et tolerogenes - Google Patents

Derives d'oligosaccharide immunosuppresseurs et tolerogenes

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Publication number
EP0588852A1
EP0588852A1 EP92911470A EP92911470A EP0588852A1 EP 0588852 A1 EP0588852 A1 EP 0588852A1 EP 92911470 A EP92911470 A EP 92911470A EP 92911470 A EP92911470 A EP 92911470A EP 0588852 A1 EP0588852 A1 EP 0588852A1
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EP
European Patent Office
Prior art keywords
group
oligosaccharide
glycoside
hydrogen
saccharide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP92911470A
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German (de)
English (en)
Inventor
Robert Ippolito
Richard H. Smith
Andre P. Venot
Mohammed A. Kashem
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Glycomed Inc
Original Assignee
Alberta Research Council
Glycomed Inc
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Filing date
Publication date
Application filed by Alberta Research Council, Glycomed Inc filed Critical Alberta Research Council
Publication of EP0588852A1 publication Critical patent/EP0588852A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention is directed to methods of employing oligosaccharide glycosides in the treatment of cell-mediated immune responses, as well as to pharmaceutical compositions containing such oligosaccharide glycosides. Specifically, the methods of the present invention are directed to methods of employing oligosaccharide glycosides related to blood group determinants in modulating (eg. suppressing) cell-mediated immune responses, including cell-mediated inflammatory responses.
  • sense a molecular signal of an apposing cell via specific binding, and biochemical mechanisms exist to translate that binding into a cellular response.
  • complex cell-surface interactions are believed to help direct processes such as binding of pathogens to target tissues 6,7 , sperm-egg binding 8 , interactions among cells in the immune system 9,10 , and recognition of cells during embryonic development 11 .
  • defects in cell-cell recognition are thought to underlie the uncontrolled cell growth and motility which characterize neoplastic transformation and metastasis 12,13 .
  • Other evidence suggests that cell-recognition processes are mediated by carbohydrate chains or glycan portions of
  • glycoconjugates 4,14-16 For example, the binding of the surface glycoconjugates of one cell to the complementary carbohydrate-binding proteins (lectins) on another cell can result in the initiation of a specific interaction.
  • lectins complementary carbohydrate-binding proteins
  • LEC-CAM proteins Lectin + EGF + complementary Regulatory Domains-Cell Adhesion Molecules. These or functionally similar proteins or lectins may play a critical role in immune responses (including inflammatory responses) through mediation of cell-cell contact and through extravasation of leucocytes 17-21 .
  • Specific carbohydrate ligands have recently been identified as part of the putative receptor structures for LEC-CAM proteins 17"21 . The structures identified include:
  • Q represents another suitably bonded sugar or sugars.
  • the present invention is directed to the discovery that low molecular weight (MW generally less than about 2000 daltons) oligosaccharide glycosides related to blood group determinants also interact with LEC-CAM proteins and/or other lectins with sufficient strength to suppress, in vivo, mammalian cell-mediated immune responses including cell-mediated inflammatory responses.
  • the present invention is also directed to the discovery that when such oligosaccharide glycosides related to blood group determinants are administered to a mammal in response to an antigen challenge, such administration induces tolerance to additional challenges from the same antigen.
  • the present invention is directed to a method of suppressing a cell-mediated immune response in a mammal which method comprises administering to said mammal an amount of an oligosaccharide glycoside related to blood group determinants effective in suppressing said immune response.
  • the immune response suppressed by this method is an inflammatory response.
  • the oligosaccharide glycoside related to blood group determinants employed in this method is further characterized as a binding-inhibitory oligosaccharide glycoside (as defined below).
  • the present invention is directed to a method of treating a cell-mediated immune response to an antigen in a mammal which method comprises administering to said mammal from about 0.5 mg/kg to about 50 mg/kg of an oligosaccharide glycoside related to blood group determinants.
  • the present invention is directed to a method of reducing sensitization of a mammal to an antigen which comprises
  • the present invention is directed to a pharmaceutical composition suitable for parenteral administration to a mammal which comprises a pharmaceutically inert carrier and an amount of oligosaccharide glycoside related to a blood group determinant effective in treating a cell-mediated immune response in said mammal.
  • FIG. 1 illustrates the increase in footpad swelling of immunized mice arising from a DTH inflammatory response measured 24 hours after challenge with 10 ⁇ g of the L1 1 1 S-Layer protein antigen wherein some of the mice have been treated at 5 hours after the challenge with 100 ⁇ g of different oligosaccharide glycosides related to blood group determinants.
  • FIG. 2 illustrates the increase in footpad swelling of immunized mice arising from a DTH inflammatory response measured 24 hours after challenge with 20 ⁇ g of the L1 1 1 S-Layer protein antigen wherein some of the mice have been treated at 5 hours after challenge with various doses of different mono- and oligosaccharide glycosides including oligosaccharide glycosides related to blood group
  • FIG. 3 illustrates secondary antibody responses (i.e., as determined by the amount of antibody measured by quantification of o-phenylenediamine O.D. at 490 nm) two weeks after primary
  • FIG. 4 illustrates the effect of an oligosaccharide glycoside related to blood group determinants, i.e., the 8-methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III, on the inflammatory DTH response in immunized mice challenged with the L111 S-Layer protein antigen wherein the mice were treated at various times before or after challenge with 100 ⁇ g of the 8-methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III.
  • an oligosaccharide glycoside related to blood group determinants i.e., the 8-methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III
  • FIG. 5 illustrates the long term (8 weeks) immunosuppression generated in immunized mice after an injection with 5 mg/kg of oligosaccharide glycosides related to blood group determinants 5 hours after challenge with 20 ⁇ g of the L111 S-Layer protein antigen on day 7.
  • FIG. 6 illustrates the long term (6 weeks) immunosuppression generated in immunized mice after an injection with varying amounts of mono- and oligosaccharide glycosides including oligosaccharide glycosides related to blood group determinants 5 hours after challenge with 20 ⁇ g of the L11 1 S-Layer protein antigen on day 7.
  • FIG. 7 illustrates the long term (10 weeks) immunosuppression generated in immunized mice after an injection with 5 mg/kg of the 8-methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III, at various times before, at and after challenge with 20 ⁇ g of the L111 S-Layer protein antigen on day 7.
  • FIG. 8 illustrates the cyclophosphamide induced restoration of a DTH inflammatory response in immunized mice previously suppressed by treatment with the 8-methoxycarbo ⁇ yioctyl glycoside of Sialyl Lewis X, Compound III.
  • FIG. 9 illustrates that the nature of the antigen used to induce the inflammatory response does not affect the ability of the 8-methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III, to regulate the DTH response.
  • FIG. 10 illustrates that the 8-methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III, can inhibit binding of U937 or HL60 to TNF ⁇ activated human umbilical vein endothelial cells (HUVECs).
  • U937 or HL60 to TNF ⁇ activated human umbilical vein endothelial cells
  • FIG. 1 1 illustrates the "core” structure of preferred
  • FIG. 12 illustrates the structures of specific oligosaccharide glycosides related to blood group determinants for use in this invention wherein R is -(CH 2 ) 8 C(O)OCH 3 .
  • FIG. 13 illustrates the structures of a couple of monosaccharide glycosides and one disaccharide glycoside used in some of the examples wherein R is -(CH 2 ) 8 C(O)OCH 3 .
  • FIG. 14 illustrates a general synthetic scheme used for the synthesis of derivatives of Neu5Ac.
  • FIG. 15 illustrates the structures of mono- and oligosaccharide glycosides 3b to 7a.
  • FIG. 16 illustrates a general reaction scheme for the synthesis of oligosaccharide glycoside 4c. as specified in Example 8 and for the synthesis of monosaccharide glycoside 37 as specified in Example 9.
  • FIG. 17 illustrates the enzymatic transfer of Neu5Ac, and of analogues thereof (collectively “sialic acids”) by the
  • FIG. 17 also illustrates the enzymatic transfer of L- fucose onto the sialylated oligosaccharide glycosides.
  • FIG. 18 illustrates the enzymatic transfer of Neu5Ac, analogues thereof (collectively “sialic acids”) by the
  • FIG. 18 also illustrates the enzymatic transfer of L-fucose onto the sialylated oligosaccharide glycosides.
  • FIG. 19 illustrates the enzymatic transfer of Neu5Ac, analogues thereof (collectively "sialic acids”) by the ⁇ Gal(1 ⁇ 4) ⁇ GlcNAc ⁇ (2 ⁇ 6')sialyltransferase to a ⁇ Gal(1 ⁇ 4) ⁇ GlcNAc- terminal structure.
  • FIG. 20 illustrates the enzymatic transfer of Neu5Ac, analogues thereof (collectively “sialic acids”) by the
  • FIG. 21 illustrates the enzymatic transfer of Neu5Ac, analogues thereof (collectively “sialic acids”) by the
  • FIGS. 22 and 23 illustrate the reaction schemes involved in the synthesis of analogues of Sialyl Lewis A by chemical modification of a sialylated hapten.
  • FIG. 24 illustrates the reaction schemes involved in the synthesis of analogues of Sialyl Lewis X by chemical
  • FIG. 25 illustrates the synthetic pathway leading to Sialyl dimeric Lewis x and internally monofucosylated derivatives thereof.
  • the nomenclature for compoound 61a is
  • ⁇ Gal(1-4) ⁇ GlcNAc(1-3) ⁇ Gal(1-4) ⁇ GlcNAc-OR sometimes called di-N- acetyliactosamin ⁇ l tetrasaccharide.
  • the hexasaccharide moiety present in compounds 65a and 65b in FIG. 1 is sometimes called VIM -2 epitope or CD-65 and 67a and 67b are called sialyl dimeric Lewis x .
  • FIG. 26 illustrates the synthetic pathway leading to the
  • FIG. 27 illustrates the increase in foot-pad swelling of immunized mice arising from a DTH inflammatory response measured 24 hours after challenge with HSV antigen, where some of the mice were treated with Sialyl Lewis X, Compound III, at the time of immunization and some of the mice were treated with Sialyl Lewis X, Compound III, 5 hours after the challenge.
  • FIG. 28 illustrates the secondary antibody responses (i.e., as determined by the amount of antibody measured by quantification of o- phenylenediamine O.D. at 490 nm) two weeks after primary
  • FIG. 29 illustrates the c ⁇ clophosphamide (CP) induced
  • FIG. 30 illustrates the effect of the 8-methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III on the inflammatory DTH response in immunized mice challenged with the OVA antigen wherein the mice were treated with Compound III five hours after challenge by a variety of different methods (IV-intravenously; IN- intranasally)
  • FIG. 31 illustrates the effect of the 8-methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III on the inflammatory DTH response in immunized mice challenged with the OVA antigen wherein the mice were treated with various doses of Compound III five hours after challenge by a variety of different methods (IV-intravenously; IN-intranasally).
  • FIG. 32 illustrates the effect of the 8-methoxycarbonyloctyl glycoside of Sialyl Lewis X, Compound III on the inflammatory DTH response in immunized mice challenged with the OVA antigen wherein the mice were treated with Compound III by a variety of different methods at the time of immunization of the mice (IV-intravenously; IN-intranasally; IM-intramuscularly)
  • FIG. 33 illustrates the effect of various oligosaccaride and monosaccharide glycosides have on the inflammatory response in the iungs of mice wherein the mice received LPS intranasally and the various compounds five hours later intravenously.
  • FIG. 34 illustrates the effect of different amounts of Sialyl LewisX and Sialyl LewisA on the lymphoproliferative response.
  • the present invention is directed at the discovery that certain low molecular weight oligosaccharide glycosides (MW less than about 2000 daltons) are effective in suppressing cell-mediated immune responses in a mammal, including cell-mediated and immune directed inflammatory responses to an antigen in a mammal (e.g., a DTH response). Additionally, treatment with these oligosaccharide glycosides also provides for induction of tolerance to the antigen in the so-treated mammal.
  • oligosaccharide glycosides MW less than about 2000 daltons
  • cell-mediated immune response in a mammal refers to those mammalian immune responses which are mediated by cell-cell interactions. Included within this term are cell-mediated inflammatory responses to an antigen such as delayed-type hypersensitivity (DTH) responses as well as cell-mediated inflammatory responses arising from myocardial infarction, virus-induced pneumonia, shock and sequelae (e.g., multiple organ failure), adult respiratory distress syndrome, and the like.
  • DTH delayed-type hypersensitivity
  • the cell-mediated immune response is a leucocyte-mediated response.
  • glycoconjugate antigens on red blood cells which serve as the basis for assigning blood into various classes according to immunological compatibility.
  • blood group determinant refers to any naturally occurring oligosaccharide segment of the nonreducing-terminal, 3-9 glycosyl residues that constitute the glycan chains of blood group substances.
  • oligosaccharide glycosides relating to a blood group determinant refer to an oligosaccharide glycoside (a) having an oligosaccharide group of from 3 to 9 saccharide units, (b) which is terminated with an aglycon group on the non-reducing sugar, and (c) wherein the oligosaccharide group is a blood group determinant (as defined above) or an analogue thereof.
  • Analogues of blood group determinants include those wherein one or more of the monosaccharide units of the blood group determinant has or have been chemically modified so as to introduce and/or remove one or more functionalities in one or more of the saccharide unit(s).
  • modification can result in the removal of an -OH functionality, the removal of saccharide unit(s), the introduction of an amine functionality, the introduction of a halo functionality, the introduction of one or more saccharide unit(s), and the like.
  • Such oligosaccharide glycosides related to blood group determinants can be represented by the formula:
  • oligosaccharide represents a carbohydrate structure of from 3 to about 9 saccharide units which oligosaccharide contains a blood group determinant or analogues thereof; Y is selected from the group consisting of O, S, > NH and a bond; and R represents an aglycon moiety of at least 1 carbon atom. Oligosaccharide glycosides related to blood group determinants are different from glycoconjugates, including blood group substances, because the aglycon moiety is neither a protein or a lipid capable of forming a micelle or other large aggregate structure.
  • the aglycone moiety, R is selected from the group consisting of -(A)-Z' wherein A represents a bond, an alkylene group of from 2 to 10 carbon atoms, and a moiety of the form -(CH 2 -CR 4 G) n - wherein n is an integer equal to 1 to 5;
  • R 4 is selected from the group consisting of hydrogen, methyl, or ethyl;
  • G is selected from the group consisting of hydrogen, oxygen, sulphur, nitrogen, phenyl and phenyl substituted with 1 to 3 substituents selected from the group consisting of amine, hydroxyl, halo, alkyl of from 1 to 4 carbon atoms and alkoxy of from 1 to 4 carbon atoms; and
  • Z' is selected from the group consisting of hydrogen, methyl, phenyl and nitrophenol and, when G is not oxygen, sulphur or nitrogen and A is not a bond, then Z' is also selected from the group consisting
  • the nitro group is reduced to an amino group which can be protected as N-trifluoroacetamido.
  • the trifluoroacetamido group is removed thereby unmasking the amino group.
  • linking arm containing sulfur is disclosed by Dahmen et al. 57 .
  • the linking arm is derived from a 2-bromoethyl group which, in a substitution reaction with thio-nucleophiles, has been shown to lead to linking arms possessing a variety of terminal functional groups such as -OCH 2 CH 2 SCH 2 SCO 2 CO 3 and
  • Rana et al. 58 discloses a 6-trifluoroacetamido)-hexyl linking arm (-O-(CH 2 ) 6 -NHCOCF 3 ) in which the trifluoroacetamido protecting group can be removed unmasking the primary amino group used for coupling.
  • linking arms include the 7- methoxycarbonyl-3,6,dioxaheptyl linking arm 59
  • R group can be an additional saccharide or an oligosaccharide containing a linking arm at the reducing sugar terminus.
  • the use of a hydrophobic group and most especially, a -(CH 2 ) 8 COOCH 3 , or -(CH 2 ) 5 OCH 2 CH CH 2 or
  • -(CH 2 ) 8 CH 2 OH group may provide for some enhancement of the acceptor properties for transfer sialic acid by this siaiyltransferase.
  • oligosaccharide glycosides related to blood group determinants effectively interfere with an immune response, particularly an
  • the oligosaccharide glycoside related to blood group determinants is further characterized as a binding-inhibitory
  • oligosaccharide glycoside i.e., an oligosaccharide glycoside related to blood group determinants which oligosaccharide glycoside binds sufficiently to a cell surface lectin so as to inhibit leucocytes from binding to another cell.
  • binding-inhibitory oligosaccharide glycosides are particularly effective in suppressing leucocyte-mediated immune responses.
  • binding-inhibitory oligosaccharide glycosides are preferred in the treatment of leucocyte-mediated immune responses.
  • lymphoproliferative experiments which measure the ability of lymphocytes to respond to an antigen can be employed to ascertain the ability of an oligosaccharide glycoside to inhibit or enhance this response.
  • An integral part of this response is the ability of lymphocytes to recognize and bind antigen-presenting cells, which recognition event triggers the proliferation of the
  • lymphocytes Such in vitro experiments are known in the art as disclosed by Ziola et al 55 .
  • other in vitro experiments which measure the ability of leucocytes to bind to the surface of cells can be employed to ascertain the ability of a candidate oligosaccharide glycoside to inhibit the ability of such cells to heterotypically or homotypically bind leucocytes to their surfaces.
  • Such in vitro experiments are well known in the art and are disclosed by Campanero et al. 26 which describes procedures for determining the homotypic binding of leucocytes to other leucocytes; and Lowe et al. 18 which describes procedures for determining the heterotypic binding of leucocytes to the surfaces of other cells.
  • the in vitro experiments are generally performed by measuring cell binding in the presence or absence (control) of the candidate oligosaccharide glycoside, e.g., at a concentration of about 10 ⁇ g/mL of a candidate oligosaccharide glycoside.
  • the extent of leucocyte binding to the cell surface is measured in both cases and candidate oligosaccharide glycosides which reduce leucocyte binding by at least about 20 percent (and preferably by at least about 30 percent and even more preferably at least about 50 percent) compared to control are deemed binding-inhibitory oligosaccharide glycosides.
  • Saccharide units (i.e., sugars) useful in the oligosaccharide glycosides related to blood group determinants employed in this invention include by way of example, all natural and synthetic derivatives of glucose, galactose, N-acetyl-glucosamine, N-acetyl-galactosamine, fucose, sialic acid (as defined below), 3-deoxy-D,L-octulosonic acid and the like.
  • all saccharide units in the oligosaccharide glycosides related to blood group determinants are In their D form except for fucose which is in its L form.
  • Preferred oligosaccharide glycosides related to blood group determinants are those which contain from 3 to 8 saccharide units especially those containing the ⁇ Gal(1 ⁇ 4) ⁇ GlcNAc or
  • R is an aglycon, preferably as defined above, each R 1 is independently selected from the group consisting of hydrogen, a saccharide and a compatible
  • each R 2 is independently selected from the group consisting of hydrogen, a saccharide and a compatible saccharide and at least one of R 1 and R 2 is a saccharide.
  • oligosaccharide glycosides related to blood group determinants include those having the and the groups where Y and R are as defined above. Even more preferred oligosaccharide glycosides related to blood group determinants are those which contain a N-acetylneuraminic acid residue or an analogue thereof particularly as the non-reducing sugar terminus of the oligosaccharide.
  • compatible saccharide refers to those substituent saccharide groups which when substituted on an existing
  • oligosaccharide glycoside related to a blood group determinant structure still permit the resulting structure to interfere with the immune response so as to reduce or inhibit the degree of immune response.
  • substitution of a particular saccharide or a combination of saccharides so alters the characteristics of the oligosaccharide glycoside related to a blood group determinant so as to render the resulting structure incapable of inhibiting an immune response, then such a saccharide substituent or combination of substituents would be deemed an incompatible substituent at least as it relates to substitution at that point on the structure of the
  • oligosaccharide glycoside related to a blood group determinant.
  • sialic acid refers to (N-acetylated)
  • Oligosaccharide glycosides including oligosaccharide glycosides related to blood group determinants, are readily prepared either by complete chemical synthesis or by chemical/enzymatic synthesis wherein glycosyltransferases are employed to effect the sequential addition of one or more sugar units onto a saccharide or an
  • oligosaccharide Chemical synthesis is a convenient method for preparing either the complete oligosaccharide glycoside; for chemically modifying a saccharide unit which can then be chemically or
  • Chemical modifications of saccharide units are well known in the art.
  • chemically modified Neu5Ac derivatives including 9-azido-Neu5Ac, 9-amino-Neu5Ac, 9-deoxy-Neu5Ac, 9-fluoro-Neu5Ac, 9-bromo-Neu5Ac, 8-deoxy-Neu5Ac, 8-epi-Neu5Ac, 7-deoxy-Neu5Ac, 7-epi-Neu5Ac, 7,8-bis-epi-Neu5Ac, 4-O-methyl-Neu5Ac, 4-N-acetyl- Neu5Ac, 4,7-di-deoxy-Neu5Ac, 4-oxo-Neu5Ac, 3-hydroxy-Neu5Ac, 3-fluoro-Neu5Ac acid as well as the 6-thio analogues of Neu5Ac are known in the art.
  • Chemical modifications of other saccharide units are also known in the art.
  • oligosaccharide glycosides are also well known in the art which methods are generally adapted and optimized for each individual structure to be synthesized.
  • the chemical synthesis of all or part of the oligosaccharide glycosides first involves formation of a glycosidic linkage on the anomeric carbon atom of the reducing sugar.
  • an appropriately protected form of a naturally occurring or of a chemically modified saccharide structure (the glycosyl donor) is selectively modified at the anomeric center of the reducing unit so as to introduce a leaving group comprising halides, trichloroacetimidate, thioglycoside, etc.
  • the donor is then reacted under catalytic
  • the saccharide glycoside can be used to effect coupling of additional saccharide unit(s) or chemically modified at selected positions or, after
  • glycos ⁇ l transferases which transfer sugar units, activated as their appropriate nucleotide donors, to specific saccharide or
  • oligosaccharide acceptors generally at the non-reducing sugar portion of the saccharide or oligosaccharide. See, for example, Toone et al. 34 .
  • glycos ⁇ ltransferases are sial ⁇ ltransferases which constitute a group of enzymes which transfer N- acetylneuraminic acid, activated as its cytidine monophosphate (CMP) derivative, to the terminal oligosaccharide structures of glycolipids or gl ⁇ coproteins.
  • CMP cytidine monophosphate
  • CMP nucleotide
  • Activation of Neu5Ac is usually done by using the enzyme CMP-sialic acid s ⁇ nthase which is readily available and the literature provides examples of the activation of various analogues of Neu5Ac such as 9- substituted Neu5Ac, 7-epi-Neu5Ac, 7,8-bis-epi-Neu5Ac, 4-O-methyl-Neu5Ac,4-deoxy-Neu5Ac, 4-acetamido-Neu5Ac, 7-deoxy-Neu5Ac, 4,7-dideoxy-Neu5Ac, and the 6-thio derivatives of Neu5Ac.
  • CMP-sialic acid s ⁇ nthase which is readily available and the literature provides examples of the activation of various analogues of Neu5Ac such as 9- substituted Neu5Ac, 7-epi-Neu5Ac, 7,8-bis-epi-Neu5Ac, 4-O-methyl-Neu5Ac,4-deoxy-Neu5A
  • the Neu5Ac analogue can be coupled to the oligosaccharide acceptor by chemical means known in the art.
  • nucleotide derivative of Neu5Ac or of an analogue thereof and the saccharide acceptor are combined with each other in the presence of a suitable sialyltransferase under conditions wherein Neu5Ac or an analogue thereof is transferred to the acceptor.
  • the saccharide acceptor employed must be one which functions as a substrate of the particular sialyltransferase employed.
  • sialyltransferases show strict specificity for one type of acceptor. It has been found that chemically modified acceptors (“artificial acceptors”), such as oligosaccharide glycosides optionally modified in the oligosaccharide portion, are tolerated in some cases by
  • ⁇ Gal(1 ⁇ 3/4) ⁇ GIcNAc ⁇ (2 ⁇ 3)sialyltransferase can transfer Neu5Ac to a terminal ⁇ Gal(1 ⁇ 4) ⁇ GlcNAc- disaccharide structure.
  • the hydroxyl groups at the 3, 4 and 6 positions of ⁇ -galactose are critical to recognition by the enzyme and accordingly chemical modification at one or more of these points can result in non-recognition by the enzyme.
  • sialyltransferases are naturally designed to transfer or donate Neu5Ac, any modification to the Neu5Ac results in the formation of an "artificial donor".
  • the art recognizes that certain sialyltransferases can tolerate some modifications to the Neu5Ac and still transfer analogues of Neu5Ac to glycoproteins or glycoiipids possessing a suitable terminal acceptor structure.
  • sialyltransferases possess sufficient recognition flexibility so as to transfer an artificial donor to an artificial acceptor. Such flexibility permits the facile synthesis of numerous sialic acid containing oligosaccharide glycosides.
  • a suitable nucleotide derivative of Neu5Ac or an analogue thereof is combined with a suitable acceptor (i.e., a
  • oligosaccharide glycoside or an oligosaccharide glycoside having terminal saccharide unit(s) on the non-reducing end which are recognized by the sialyltransferase) in the presence of the sialyltransferase under conditions wherein Neu5Ac or an analogue thereof is transferred to the acceptor.
  • Suitable conditions include the addition of the appropriate sialyltransferase to a mixture of the saccharide acceptor and of the CMP-derivative of the sialic acid in a appropriate buffer such as 0.1 M sodium cacodylate in appropriate conditions of pH and temperature such as at a pH of 6.5 to 7.5 and a temperature between 25 and 45°C, preferably 35-40°C for 12 hours to 4 days.
  • the resulting oligosaccharide can be isolated and purified using conventional methodology comprising HPLC, gel-, ion exchange-, reverse-phase- or adsorption chromatography.
  • sugars can be transferred onto a saccharide or oligosaccharide structure by use of appropriate glycosyltransferases in a manner similar to that described above for transfer by
  • sialyltransferases as well as other glycosyltransferases are well known in the art and are described in
  • oligosaccharide glycosides affect the cell mediated immune response in a number of ways. Oligosaccharide glycosides can inhibit the ability of the immune response to become educated about a specifc antigen when the oligosaccharide glycoside is administered simultaneously with the first exposure of the immune system to the antigen. Also, oligosaccharide glycosides can inhibit the effector phase of a cell-mediated immune response (eg., the inflammatory component of a DTH response) when administered after second or later exposures of the immune system to the antigen. Additionally, oligosaccharide glycosides can induce tolerance to antigens when administered at the time of second or later exposures of the immune system to the antigen.
  • a cell-mediated immune response eg., the inflammatory component of a DTH response
  • the suppression of the inflammatory component of the immune response by oligosaccharide glycosides related to blood group determinants is believed to require the initiation of a secondary immune response (i.e., a response to a second exposure to antigen).
  • the oligosaccharide glycoside related to a blood group determinant is generally administered to the patient at least about 0.5 hours after an inflammatory episode, preferably, at least about 1 hour after, and most preferably, at least about 5 hours after an inflammatory episode or exacerbation.
  • Oligosaccharide glycosides related to blood group determinants are effective in suppressing cell-mediated immune responses to an antigen (eg. the inflammatory component of a DTH response) when administered at a dosage range of from about 0.5 mg to about 50 mg/kg of body weight, and preferably from about 0.5 to about 5 mg/kg of body weight.
  • an antigen eg. the inflammatory component of a DTH response
  • the specific dose employed is regulated by the particular cell-mediated immune response being treated as well as by the judgment of the attending clinician depending upon factors such as the severity of the adverse immune response, the age and general condition of the patient, and the like.
  • the oligosaccharide glycosides related to blood group determinants are generally administered parenterally, such as intranasally, intrapulmonarily, transdermally and intravenously, although other forms of administration are
  • the suppression of a cell-mediated immune response eg. the inflammatory component of a DTH response
  • the suppression of a cell-mediated immune response is reduced by at least about 10% as opposed to control measured 24 hours after administration of the challenge to the mammal and 19 hours after administration of the oligosaccharide glycoside as per this invention.
  • administration of the oligosaccharide glycoside related to a blood group determinant also imparts a tolerance to additional challenges from the same antigen.
  • re-challenge by the same antigen weeks after administration of the oligosaccharide glycoside related to a blood group determinant results in a significantly reduced immune response.
  • reducing sensitization means that the compound, when administered to a mammal in an effective amount along with a sufficient amount of antigen to induce an immune response, reduces the ability of the immune system of the mammal to become educated and thus sensitized to the antigen administered at the same time as the compound.
  • an "effective amount" of the compound is that amount which will reduce sensitization (immunological education) of a mammal to an antigen administered simultaneously as determined by a reduction in a cell-mediated response to the antigen such as DTH responses as tested by the footpad challenge test.
  • the reduction in sensitization will be at least about 20% and more preferably at least about 30% or more.
  • oligosaccharide glycosides related to blood group determinants are effective in reducing sensitization when administered at a dosage range of from about 0.5 mg to about 60 mg/kg of body weight, and preferably from about 0.5 mg to about 5 mg/kg of body weight.
  • sensitization is regulated by the sensitization being treated as well as the judgement of the attending clinician depending upon the age and general condition of the patient and the like.
  • “Simultaneous" administration of the compound with the antigen with regard to inhibiting sensitization means that the compound is administered once or continuously throughout a period of time within 3 hours of the administration of an antigen, more preferably the compound is administered within 1 hour of the antigen.
  • compositions suitable for use in the parenteral administration of an effective amount of an oligosaccharide glycoside related to a blood group determinant comprise a pharmaceutically inert carrier such as water, buffered saline, etc. and an effective amount of an oligosaccharide glycoside related to a blood group determinant so as to provide the above-noted dosage of the oligosaccharide glycoside when administered to a patient. It is " contemplated that suitable pharmaceutical compositions can
  • compositions can include oral compositions, transdermal compositions or bandages etc., which are well known in the art.
  • BSA bovine serum albumin
  • DTH delayed-type hypersensitivity
  • Examples 1-19 illustrate the synthesis of numerous oligosaccharide glycosides whereas Examples 20-37 illustrate the suppression of cell-mediated immune responses to an antigen by administration of an oligosaccharide glycoside related to blood group determinants and the induced tolerance to later challenges with the same antigen.
  • Examples 1-13 the oligosaccharide glycosides recited are referred to by Arabic numerals which are depicted in figures 14-24 whereas in Examples 20-35, the
  • oligosaccharide glycosides are referred to by Roman numerals which are depicted in figures 11-13.
  • Examples 1-13 pre-coated plates of silica gel (Merck, 60-F 254, ) were used for analytical t.l.c. and spots were detected by charring after spraying with a 5% solution of sulfuric acid in ethanol.
  • Silica gel 60 (Merck, 40-63 ⁇ m) was used for column chromatography.
  • latrobeads were from latron (Order No. 6RS-8060).
  • Millex-GV filters (0.22 ⁇ m) were from Millipore.
  • C 18 Sep-Pak cartridges and bulk C 18 silica gel were from Waters Associates.
  • reaction mixtures were processed by dilution with dichloromethane and washing with a dilute solution of sodium bicarbonate followed by water. After drying over magnesium sulfate, the solvents were removed by evaporation under vacuum with a bath temperature of 35 °C or lower when necessary.
  • FIG. 14 illustrates a general synthetic scheme used for the synthesis of derivatives of Neu5Ac.
  • Compounds referred to by underlined Arabic numerals in Examples 1-4 below are depicted Table I and in FIG. 14.
  • dichloromethane 13 mL was added to the mixture of benzyl alcohol (5.0 mL, 48.2 mmol), molecular sieves 4A (18.5 g, crushed), dry silver carbonate (4.2 g, 15.2 mmol) in dichloromethane (8 mL). The mixture was stirred in the dark for 4 days, diluted with
  • Propionic anhydride (0.12 mL, 0.94 mmol) was then syringed into a suspension of the above product in a mixture of dry methanol (1.5 mL) and triethylamine (0.2 mL) which was stirred at 0°C.
  • the above product (0.091 g, 0.169 mmol) was heated for 4 hours at 40 °C in 70% aqueous acetic acid. The mixture was co-evaporated with toluene in vacuo. The dry residue was dissolved in dry methanol and stirred for 2 hours at 22°C in the presence of sodium metaperiodate (0.069 g, 0.276 mmol). The mixture was filtered through a pad of Celite which was washed with methanol. The combined filtrate was stirred at 0°C for 26 minutes in the presence of sodium borohydride (0.036 g, 0.95 mmol).
  • CMP-sialic acid synthase was extracted from calf brain and partially purified at 4°C by a slight modification of the original procedure of Higa et al. 40 Routinely, ⁇ 200 g of brain tissue were homogenized in a Cuisinart blender (three 30 second bursts with 1 minute intervals) with 400 mL of 25 mM Tris/HCI, pH 7.5, 10 mM magnesium chloride, 10 mM sodium chloride, 2.5 mM dithioerythritol, 0.5 mM phenylmethylsulfonyl fluoride. The homogenate was stirred for 1 hour and then centrifuged at 23,000 ⁇ g for 15 minutes.
  • the supernatant was decanted and the pellets were extracted once again with 200 mL of the same buffer as above.
  • the supernatants were combined and centrifuged at 28,000 ⁇ g for 15 minutes.
  • the supernatant was filtered through glass wool to give the crude extract
  • the crude extract was stirred and solid ammonium sulfate was added to 35% saturation (208 g/L) over a period of 15 minutes.
  • the solution was stirred for an additional 15 minutes, kept on ice for 1 hour and centrifuged at 28,000 ⁇ g for 30 minutes. The precipitate was discarded and the supernatant was stirred and adjusted to 60% saturation by the addition of solid ammonium sulfate (163 g/L) over 15 minutes. After an additional 15 minutes of stirring, the suspension was left on ice overnight and then centrifuged as above. The resultant pellets were washed with 150 mL of 60% ammonium sulfate solution to remove the co-precipitates.
  • the washed pellets contain 70-80 U of enzyme with a specific activity of 0.08 U/mg protein.
  • the enzyme was assayed as described by Kean et al. 41 , with one unit of enzymatic activity defined as one //mol of product formed per minute at 37°C.
  • the enzyme present in the pellet could be stored for several weeks in the cold room. Before using the enzyme for synthesis, the pellets were suspended in a minimal volume of 50 mM Tris/HCI, pH
  • the CMP-derivatives of sialic acid analogues were synthesized as noted above and purified by a modification of the reported procedures of Higa et al. 40 and Gross et al. 42
  • 7-d-Neu5Ac 1d (Table 1 , 20 mg, 69 //mol) was activated by using 15 U of the above dialyzed enzyme for 5-6 hours at 37°C in 12 mL of the activation buffer in the presence of four fold excess of cytidine triphosphate.
  • the conversion of the sialic acid analogues was estimated by the usual thiobarbituric acid assay for sialic acid after reduction with sodium borohydride as per Kean et al. 43
  • the product was extracted with cold acetone as per Gross et al. 42
  • Examples 6-7 illustrate the synthesis of oligosaccharide glycosides.
  • the structure of 3b to 7 a are illustrated in FIG. 15.
  • Oligosaccharide glycosides 4b, 5b, 5f, 6a, and 7a. were synthesized according to the procedures of Lemieux et al. 43 , Lemieux et al. 44 ,
  • Oligosaccharide glycosides 4d and 5d were synthesized following the procedure reported for the synthesis of oligosaccharide glycosides 4b and 5b, but by replacing the 8-methoxycarbonyloctyl by methanol.
  • Oligosaccharide glycosides 5e and 5g were synthesized according to the procedures of Paulsen et al. 45 and Alais et al. 48 but replacing the methanol by 8-methoxycarbonyloctanoI. In all cases, the oligosaccharide glycosides were purified by chromatography on latrobeads with the appropriate solvent mixtures and the recovered materials chromatographed on BioGel P2 or Sephadex LH20 and eluted with water. The recovered materials were lyophilized from water and the products further dried in vacuo over phosphorus pentoxide.
  • Example 6 ⁇ Synthesis of 9-Hydroxynonyl 2-acetamido-2- deoxy-[ ⁇ -D-galactopyranosyl-(1-3)-O-]- ⁇ -D- glucopyranoside 4a.
  • acetimidate 34 (1.70 g, 3.50 mmol) and molecular sieves (0.500 g, crushed in a 1 :1 mixture of toluene and dichloromethane (30 mL) cooled to -20°C. The mixture was stirred at -20°C for 0.5 hours and slowly brought to 0°C in 1 hour. T.l.c. (1 :1 hexane and ethyl acetate) indicated the completion of the reaction. Some triethylamine was added and after dilution with methylene chloride and filtration, the solvents were worked up in the usual manner.
  • the starting material 32 (0.300 g, 0.689 mmol) was
  • Sialic acids activated as their CMP-derivatives (as set forth in Examples 1-5 above), were transferred onto synthetic oligosaccharide structures containing ⁇ Gal(1-3) ⁇ GlcNAc-, ⁇ Gal(1-4) ⁇ GlcNAc-, ⁇ Gal(1- 3) ⁇ GalNAc-, and ⁇ Gal(1-4) ⁇ Glc- terminal sequences by using three mammalian sialyl-transferases (Examples 10a-e).
  • the sialyl-transferases Examples 10a-e.
  • ⁇ Gal(1-3/4) ⁇ GlcNAc- ⁇ (2-3)sialyltransferase (EC 2.4.99.5) and the ⁇ Gal(1-4) ⁇ GlcNAc- ⁇ (2-6)sialyltransf erase (EC 2.4.99.1 ) from rat liver were purified to homogeneity by affinity chromatography according to the procedure of Mazid et al. 49 , which is incorporated herein by reference on a matrix obtained by covalently linking the hapten ⁇ Gal(1-3) ⁇ GlcNAcO(CH 2 ) 8 CO 2 H 43 (Chembiomed Ltd., Edmonton, Canada) to activated Sepharose by methods known in the art.
  • oligosaccharide (5-20 mg) was incubated with the selected CMP-sialic acids (5-20 mg) in the presence of the appropriate sialyltransferase
  • sialyloligosaccharide 7-d- ⁇ Neu5Ac(2-6) ⁇ Gal ⁇ 1- 4) ⁇ GlcNAc-O-(CH 2 ) 8 -COOCH 3 13d.
  • reaction mixture was diluted to 10 mL and passed onto three Sep-Pak C 18 cartridges, conditioned as suggested by the manufacturer. Each cartridge was washed with water (4 x 5 mL) and then with methanol (3 ⁇ 5 mL). The methanol eluate was evaporated to dryness in vacuo and the residue was dissolved in a 65:35:3 mixture of chloroform, methanol and water (0.5 mL - solvent I) and applied on to a small column of latrobeads (500 mg) equilibrated in the same solvent.
  • Sialylated analogues of the type I and II oligosaccharides can be further fucosylated by the human milk
  • ⁇ GIcNAc ⁇ (1-3/4)fucosyltransferase The enzyme was purified from human milk according to the methodology using affinity
  • oligosaccharides was carried out by a modification of the procedures of Palcic et al. s ⁇
  • the fucosylated structure 9-N 3 - ⁇ Neu5Ac(2-3) ⁇ Gal(1-3)-[ ⁇ -L-Fuc(1-4) - ⁇ GlcNAc-O-(CH 2 ) 8 -CH 2 OH 17b was synthesized by incubating GDP-fucose (2.5 mg) and 9-N 3 - ⁇ Neu5AC(2-3) ⁇ Gal(1-3) ⁇ GIcNAc-O-(CH 2 ) 8 -CH 2 OH 8b (1.7 mg) with affinity purified ⁇ GIcNAc ⁇ (1-3/4)fucosyltransferase (4.6 mU) in 1.3 mL of 100 mM sodium cacodylate (pH 6.5), 10 mM manganese chloride, 1.6 mM ATP, 1.6 mM sodium azide.
  • Examples 10a- 10e are as follows:
  • Example 10a This example refers to the transfer of modified sialic acids such as 1a-g to the 3-OH of a terminal ⁇ Gal of acceptors possessing a ⁇ Gal(1-3) ⁇ GlcNAc- (Lewis 0 or Type I) terminal structure such as 4a and 4b by a sialyltransferase such as the ⁇ Gal(1-3/4) ⁇ GlcNAc ⁇ (2-3)sialyltransferase from rat liver following the experimental procedure reported above.
  • the 1 H-n.m.r. data of the reaction products, which were purified as indicated previously, are reported (Tables 3 and 4).
  • Example 10b This example refers to the transfer of modified sialic acids such as 1b and 1c to the 3-OH of the terminal ⁇ Gal of acceptors possessing a ⁇ Gal(1-4) ⁇ GlcNAc- (LacNAc or Type II) terminal structure such as 5i# b, sLfl by a sialyltransferase such as that used in 10a.
  • dimethylsulfoxide 5% volume
  • the 1 H-n.m.r. data of the reaction products, which were purified as indicated previously, is reported (Tables 6 and 8). The reaction mixture was worked up in the manner described previously.
  • Example 10c This example refers to the transfer of modified sialic acids such as le to the 3-OH structure of the terminal ⁇ Gal of acceptors possessing a ⁇ Gal(1-4) ⁇ Glc- (lactose) terminal structure such as 6a by a sialyltransferase such as that used in Example 10a following the same experimental procedure.
  • the 1 H-n.m.r. data of the reaction products, which were purified as indicated previously, is reported (Table 6).
  • Example 10d This example refers to the transfer of modified sialic acids such as 1 b - h to the 6-OH of the terminal ⁇ Gal of acceptors possessing a ⁇ Gal(1-4) ⁇ GlcNAc- (LacNAc or Type II) terminal unit such as 5b, d-g by a sialyltransferase such as the ⁇ Gal(1-4) ⁇ GlcNAc ⁇ (2-6)sialyltransferase reported previously.
  • the 1 H-n.m.r. data of the reaction products, which were purified as indicated previously, is reported (Tables 7 and 8).
  • Example 10e This example refers to the transfer of modified sialic acids such as 1c to the 3-OH of the terminal ⁇ Gal of acceptors possessing a ⁇ Gal(1-3) ⁇ GalNAc- ("T") terminal unit such as 7 a by a sialyltransferase such as the ⁇ Gal(1-3) ⁇ GalNAc ⁇ (2-3)sialyltransferase (Genzyme) following the experimental procedure reported previously.
  • T ⁇ Gal(1-3) ⁇ GalNAc-
  • FIGs. 22-24 illustrate the reaction schemes involved in the preparation of these analogues and provide structures for the prepared analogues which are identified by an underlined arabic numeral.
  • the starting trisaccharide 8a (1.3 mg) was stirred for 24 hours at +4°C in 1.7 mL of a solution 0.05 M in sodium acetate and 0.010 M in sodium periodate. The excess of sodium periodate was then destroyed by addition of some ethylene glycol. Sodium borohydride (20 mg) was then added and the stirring was continued for 24 hours at 4°C. The pH of the reaction mixture was then brought to 6 by addition of acetic acid and the solvents were co-evaporated with methanol. The residue was dissolved in water (1 mL) and run through a Sep-Pak cartridge which was further washed with water followed by methanol.
  • Trisaccharide 8m was enzymatically fucosylated following the procedure reported in Example 10 and the product purified in the same manner. T.l.c. of the recovered crude material indicated that the transformation of 8jm was almost complete. Purification gave 17m (0.5 mg); 1 H-n.m.r.: see Table 5 above.
  • Acetic anhydride (about 0.2 mg) in methanol (10 ⁇ l) was added to a solution of 8j (about 1 mg) in a 1 :1 solution of 0.002 N sodium hydroxide and methanol (0.300 mL) at 0°C. T.l.c. (solvent as above) indicated a complete reaction and the solvents were then evaporated. The residue was dissolved in water (2 mL) and applied to a Sep-Pak cartridge. The cartridge was washed with water and the product eluted with methanol giving the trisaccharide 8k (about 1 mg); 1 H-n.m.r.: see Table 3 above.
  • Trisaccharide 8k was enzymatically fucosylated following the procedure reported in Example 10 and the product purified in the same manner. T.l.c. of the recovered crude material indicated that the transformation of fik was almost complete. Purification gave 17k
  • Tetrasaccharide 18a (0.003 g) was applied on Dowex
  • methanol water providing the methyl ester of compound 18a (0.025 g): 1 H-n.m.r.: 5.099 (d, 1H, J 1 ,2 3.75Hz, H-1 ⁇ FUC), 4.517 (d, 2H, J 1,2 7.5Hz, H-1 ⁇ Gal and ⁇ GIcNAc), 3.866 and 3.683 (2s, CO 2 CH 3 ), 2.781 (dd, 1 H, J 3ax,3e q 12.5Hz, J 3eq,4 4.5Hz, H-3eq Neu5Ac), 2.032 and 2.018 (2s, 6H, 2 NAc), 1.913 (dd, 1H, J 3ax,4 12.5Hz, H-3ax Neu5Ac),
  • analogue of blood group determinants also posess immunogenic and tolerogenic properties.
  • the analogue of the blood group determinant employed is CD65, which is a Sialyl Lewis X derivative having a ⁇ Gal(1-4) ⁇ GlcNAc-OR disaccharide glycoside attached to the reducing sugar of the Sialyl Lewis X. See further U.S. Serial No. 07/771 ,259, filed October 2, 1991 , entitled
  • such a compound is an analogue of blood group determinants because Sialyl Lewis X is a blood group determinant, as defined herein.
  • the rat liver ⁇ Gal(1-3/4) ⁇ GlcNAc ⁇ (2-3)sialyltransferase was purified by affinity chromatography 49 on a matrix obtained by covalently linking the hapten ⁇ Gal(1-3)yffGlcNAcO(CH 2 ) 8 CO 2 H 43
  • the enzymatic sialylations were carried out at 37° C in a plastic tube using a sodium cacodylate buffer (50 mM, pH 6.5) containing Triton CF-54 (0.5%), BSA (1 mg/mL) and calf intestine alkaline phosphatase. 50 The final reaction mixtures were diluted with H 2 O and applied onto C 18 Sep-Pak cartridges as reported. 51 After washing with
  • the ⁇ GlcNAc ⁇ (1-3/4)fucosyltranferase was purified from human milk, as reported. 51
  • the enzymatic reactions were carried out at- 37 °C in a plastic tube using a sodium cacodylate buffer (100 mM, pH 6.5), MnCI 2 (10 mM), ATP (1.6 mM), NaN 3 (1.6 mM).
  • the reaction products were isolated and purified as indicated above.
  • GDP-fucose as employed below is preferably prepared by the method described in U.S. Serial No. 07/848,223, filed March 9, 1992 and entitled “Chemical Synthesis of GDP-fucose", which is
  • Tetra-n-butylammonium hydroxide (40% aq. w/w, about 150g) was added dropwise to a solution of phosphoric acid (85% aq, w/w,
  • hydrochloric acid The solution was slowly absorbed onto a column of ion exchange resin Dowex 2 X 8 [200-400 mesh, 5 ⁇ 45 cm, bicarbonate form which had been prepared by sequential washing of the resin with methanol (800 mL), water (1200 mL), ammonium bicarbonate (1 M, 1600 mL) and water (1200 mL)]. Water (1000 mL) was then run through the column followed by a solution of ammonium bicarbonate (0.5 M, 2.3 mL/minute, overnight). The eluate was collected in fractions (15 mL) and the product detected by charring after spotting on a tic plate.
  • Dowex 2 X 8 [200-400 mesh, 5 ⁇ 45 cm, bicarbonate form which had been prepared by sequential washing of the resin with methanol (800 mL), water (1200 mL), ammonium bicarbonate (1 M, 1600 mL) and water (1200 mL)]. Water (1000 mL) was then run through the column followed by
  • Guanosine 5'-( ⁇ -1-fucopyranosyl)-diphosphate was prepared from ⁇ -L-fucopyranosyI-1-phosphate using two different art recognized procedures as set forth below: PROCEDURE #1 ⁇ -L-fucopyranosyl-1-phosphate and guanosine 5'-mono-phosphomorpholidate (4-morpholine-N,N'-di-cyclohexyl-carboxamidine salt, available from Sigma, St. Louis, Missouri, "GMP-morpholidate”) were reacted as described in a recent modification 64,86 of Nunez's original procedure 65 .
  • tri-n-octylamine (0.800g, available from Aldrich Chemical Company, Milwaukee, Wisconsin) was added to a mixture of ⁇ -L-fucopyranosyl-1 -phosphate (triethyl-ammonium salt, 1.00g, about 2.20 mmol) in dry pyridine (10 mL) under nitrogen the solvent removed in vacuo. The process was repeated three times with care to allow only dry air to enter the flask.
  • GMP morpholidate (2.4g, about 3.30 mmol) was dissolved in a 1 :1 mixture of dry dimethylformamide and pyridine (10 mL). The solvents were
  • Fractions 59 to 86 which contained guanosine 5'-( ⁇ -1-fucopyrariosyl)- diphosphate (Rf -0.62), also showed a narrow U.V. active spot (Rf ⁇ 0.57). Fractions 59 to 86 were pooled and freeze-dried
  • Fractions 29 to 45 and 47 to 57 were separately pooled and freeze-dried providing recovered ⁇ -L-fuco-pyranosyl-1-phosphate (0.264g and 0.223g, respectively, in which the second fraction contains some impurities). Occasionally, pooling of appropriate fractions provided some amount of guanosine 5'-( ⁇ -1-fucopyranosyl)-diphosphate in good purity ( 1 H-n.m.r.). Generally, all the material enriched in guanosine 5'-( ⁇ -1-fuco-pyranosyl)-diphosphate was dissolved in a minimum amount of water and run on the same column which had been regenerated by washing with large amounts of methanol followed by water.
  • the ⁇ -L-fucopyranosyI-1-phosphate (triethyl-ammonium salt, 0.528g, about 1.18 mmol) was dissolved in dry pyridine (20 mL) and the solvent removed in vacuo. The process was repeated three times with care to allow only dry air to enter the flask. GMP-morpholidate (1.2g, 1.65 mmol) and pyridine (20 mL) were added into the reaction flask, the solvent evaporated in vacuo and the process repeated three times as above. Pyridine (20 mL) was added to the final residue and the heterogeneous mixture was stirred for 3 to 4 days at room
  • Clostridium Perfringens neuraminidase immobilized on agarose (Sigma Chemical Company, St. Louis, MO, 1 U) in a buffer of sodium
  • Examples 20-34 illustrate the immunosuppressive properties of oligosaccharide glycosides related to blood group determinants.
  • the oligosaccharide glycosides employed are illustrated in FIGs. 12 and 13 which employ Roman numerals to identify the structure of these compounds.
  • DTH inflammatory responses were measured using the mouse footpad swelling assay as described by Smith and Ziola 22 . Briefly, groups of Balb/c mice were immunized with 10 ⁇ g of the L111 S- Layer protein, a bacterial surface protein 23 from Clostridium
  • thermohydros ⁇ /furicum L111-69 which has been shown to induce a strong inflammatory DTH response. Seven days later, each group of mice was footpad-challenged with 10 ⁇ g of L-111 S-Layer protein.
  • the resulting inflammatory footpad swelling was measured with a Mitutoyo Engineering micrometer 24 hours after challenge.
  • mice received 100 ⁇ g of
  • PBS phosphate- buffered saline
  • mice were subjected to primary immunization and challenge with L111 -S-Layer protein as described under Example 20, above.
  • groups were injected intravenously with 100 ⁇ l solutions containing 10, 25, 50, 75, or 100 ⁇ g of oligosaccharide glycoside related to blood group determinant III depicted in FIG. 12, or with PBS.
  • the DTH responses for each dose group were measured 24 hours after challenge and are shown in FIG. 2. While the groups receiving PBS or 10 ⁇ g of oligosaccharide glycoside III showed essentially the same extent of footpad swelling as PBS-treated controls, the groups receiving 25, 50, 75 or 100 ⁇ g of oligosaccharide glycoside III displayed reduced footpad swelling (78,
  • Antibody titers were determined using a solid phase enzyme immunoassay (EIA) as described by Ziola et al 24 . Briefly, 2 ⁇ g of EIA
  • L1 1 1-S-Layer protein was added per well of a Maxisorb EIA plate (Flow Laboratories, Inc., McLean, VA). Following incubation at room temperature overnight, unabsorbed antigen was removed by inverting the wells. Each well then received 200 ⁇ l of various dilutions of mouse serum prepared in phosphate-buffered saline containing 2% (w/v) bovine serum albumin and 2% (v/v) Tween 20. After 1 hour at room temperature, the solutions were removed by inverting the wells, and the wells washed four times with distilled, de-ionized water at room temperature. Horse-radish peroxidase-conjugated, goat anti- mouse immuno-globulin antibodies were then added to each well (200 ⁇ l of a 1 :2000 dilution prepared in the phosphate-buffered
  • FIG. 3 graphically illustrates the titers determined with six dilution series of sera from the L111 -immunized and challenged mice which were treated with oligosaccharide glycosides lll-VII depicted in FIG. 12 and examined for footpad swelling as described in Example 20 above.
  • the dilution curves shown in FIG. 3 indicate that the
  • mice Groups of Balb/c mice, immunized and challenged with L111 S-Layer protein as described in Example 20, were injected with a solution of 100 ⁇ g of oligosaccharide glycoside related to blood group determinant III, depicted in FIG. 12, in PBS (100 ⁇ l) at different time points relative to the time of antigen challenge.
  • PBS 100 ⁇ g of oligosaccharide glycoside related to blood group determinant III, depicted in FIG. 12, in PBS (100 ⁇ l) at different time points relative to the time of antigen challenge.
  • One group received oligosaccharide glycoside ill one hour prior to the antigen challenge; another, immediately after challenge, the third group one hour after challenge, and the fourth group 5 hours after challenge.
  • a control group was included which received PBS (100 ⁇ L) immediately after challenge.
  • Examples 20-23 above establish that treatment with an effective amount of an oligosaccharide glycoside related to blood group determinants after challenge by an antigen suppresses an immune response to the antigen (i.e., a DTH response), in as much as the level of inflammation measured 24 hours after challenge, is reduced by at least 20% in animals treated with an oligosaccharide glycoside related to blood group determinant as opposed to the level of inflammation exhibited by the control animals.
  • mice treated with oligosaccharide glycosides related to blood group determinants III- VII in Example 20 were re-challenged with L111 S-Layer protein 8 weeks after primary immunization.
  • oligosaccharide glycoside IV 69%; oligosaccharide glycoside V, 78%; oligosaccharide glycoside VI, 78%; oligosaccharide glycosdfide VII, 69%.
  • FIG. 13 were rechallenged six weeks after primary immunization. Footpad swelling similar to that of PBS-treated controls was observed with those mice that had been treated with saccharides VII I and IX, and disaccharide X, 5 hours after the first challenge. Mice originally treated with 10-100 ⁇ g of IlI showed footpad swelling that ranged from 90 to 65% of that displayed 24 hours after the first challenge. iii. The identical groups of mice which had been treated in Example 22 with 100 ⁇ g of oligosaccharide glycoside III at 1 hour before first challenge, or 5 hours after first challenge, were rechallenged with antigen 10 weeks after primary immunization.
  • mice treated before or shortly after challenge were the same as that of PBS-treated mice, whereas those mice originally treated 1 hour or 5 hours after challenge showed only about 66% of the values observed for PBS-treated controls.
  • FIGs. 5-7 demonstrate that oligosaccharide glycosides related to blood group determinants impart tolerance to challenges with the same antigen for at least 10 weeks after treatment.
  • this example employs immunized mice which have been previously suppressed and tolerized to DTH inflammatory responses by treatment with the 8-methoxycarbonyloctyl glycoside of
  • mice Fourteen days after immunization, the mice were injected with 200 mg/kg of CP and then 17 days after immunization, the mice were challenged with 20 ⁇ g of L111 S-Layer protein. 24 hours after the challenge, the extent of the DTH response was ascertained by measuring (mm -1 ) the increase in footpad swelling.
  • FIG. 8 illustrates that injection with CP prior to challenge with the L1 11 S-Layer protein restores the DTH inflammatory response in mice that have previously under-gone immunosuppressive treatment with the 8-methoxycarbonyloctyl glycoside of Compound III.
  • Example 20 Mice were immunized as outlined in Example 20 with S-Layer L11 1 , herpes simplex virus 1 (HSV 1 ) and cationized bovine serum albumin (Super CarrierTM, Pierce, Rockford, 1L). As shown in FIG. 9, the nature of the antigen used to induce the inflammatory response does not appear to affect the ability of Compound III to regulate this response.
  • HSV 1 herpes simplex virus 1
  • Super CarrierTM Pierce, Rockford, 1L
  • Vascular Endothelium This example examines whether the synthetic blood group determinant related to oligosaccharide glycoside III could inhibit ELAM- 1 dependent cell adhesion to activated vascular endothelium.
  • FIG. 10 sets forth the results of this example illustrates that
  • Compound III inhibits ELAM-1 dependent binding to the HUVECs.
  • Example 20-26 The data in Examples 20-26 above establish the effectiveness of oligosaccharide glycosides related to blood group determinants in treating immune responses to an antigen and in inducing tolerance to the antigen in a mammal (mice). In view of the fact that the immune system of mice is a good model for the human immune system, such oligosaccharide glycosides will also be effective in treating human immune responses. This is borne out by the fact that Example 27 establishes that an oligosaccharide glycoside related to blood group determinant inhibits ELAM-1 dependent binding to the HUVEC.
  • mice 100 ⁇ l PBS intramusclularly at the same site. Seven days later, the mice were footpad
  • mice The fourth group of mice was immunized
  • the extent of the DTH inflammatory response was measured 24 hours after challenge by measuring footpad swelling with a Mitutoyo
  • FIG. 27 shows the degree of footpad swelling observed.
  • mice are those mice which receive compound in addition to the antigen.
  • Untreated Mice are those mice which do not receive compound. Background swelling is that level of swelling observed in mice immunized with PBS alone without antigen or compound and challenged with antigen.
  • mice injected with SleX at the same time as and site of immunization with HSV showed a 88% reduction in footpad swelling compared to that of mice immunized with HSV and challenged with HSV.
  • Mice injected with SleX 5 hours after the footpad challenge with HSV showed an approximately 86.7% reduction in footpad swelling compared to that of mice immunized with HSV and
  • results of this example support previous examples which show that oligosaccharide glycoside III can suppress an immune response to an antigen if given to mice 5 hours after challenge by the antigen.
  • This example also shows that oligosaccharide glycoside III given to mice at the time of immunization can inhibit sensitization of the immune system to the antigen.
  • SleX compound III
  • Example 29 Effect of Sialyl LeX on the Antibody Response to HSV Four groups of mice were treated as described in Example 28.
  • Antibody titres were determined as described in Example 22 except HSV antigen was used in place of L1 11 S-Layer protein.
  • FIG. 28 graphically illustrates the titres determined with six dilution series of sera from the groups of immunized mice as described in Example 28. The results of the first two groups correlated with the results obtained in Example 22 for the L111 S-Layer Protein.
  • mice with SleX five hours after challenge did not affect the antibody response.
  • mice treated with SleX at the time of immunization showed significant reduction in the antibody response to the HSV antigen.
  • SleX compound III
  • suppressor cells can be removed by treatment of mice with cyclophosphamide (CP).
  • CP cyclophosphamide
  • this example employs immunized mice which have been previously suppressed and tolerized to DTH inflammatory responses by treatment with L1 1 1 S-Layer antigen.
  • One group of Balb/c mice were immunized with 20 ⁇ g /mouse of the L111 S-Layer protein. Seven days later, this group of mice was footpad-challenged with 20 ⁇ g of L111 S-Layer protein.
  • the second group of mice were immunized with 20 ⁇ g of the L111 protein and 100 ⁇ g of SleX at the same site and seven days later were footpad challenged with 20 ⁇ g of L111.
  • mice were injected with 200 mg/kg of CP interperitoneally two days before immunization. This group was then immunized and challenged as described for group two.
  • the fourth group of mice were immunized with 20 ⁇ g/mouse of L11 1 and 100 ⁇ g /mouse of the T-disaccharide at the same site and footpad challenged as described for group one.
  • Group five was immunized 100 ⁇ l of PBS and then footpad challenged as described for group one.
  • Example 29 The results are presented in FIG. 29. These results confirm the results discussed in Example 28 that treatment of mice with SleX at the time of immunization can suppress the immune response to an antigen. Furthermore, this Example shows that the suppression of the immune response by treatment with SleX at the time of immunization can be eliminated by cyclophosphamide treatment before immunization suggesting the involvement of cyclophosphamide sensitive suppressor
  • mice were footpad-challenged with 20 ⁇ g of OVA in 20 ⁇ l of PBS. The resulting inflammatory footpad swelling was measured with a Mitutoya
  • SleX compound III
  • mice are anethesitized with Metofane (Pitman-Moore Ltd., Mississauga,
  • mice age 8-12 weeks, weight about 20-25 g, were immunized with 100 ⁇ g of OVA (Albumin Chicken Egg, Sigma) and 20 ⁇ g of DDA in 100 ⁇ l of PBS, intramuscularly into the hind leg muscle of the mouse. Seven days after immunization, the mice were footpad challenged with 20 ⁇ g of OVA in 20 ⁇ l of PBS.
  • OVA Albumin Chicken Egg, Sigma
  • mice were either given intravenously 100 ⁇ g /mouse of SleX in 200 ⁇ l of PBS or 200 ⁇ l PBS only or given intranasally 100 ⁇ g /mouse of SleX in 50 ⁇ l of PBS or 50 ⁇ l PBS only at which procedure the mice were under light anesthesia.
  • the footpad swelling was measured 24 hours later with a Mitutoyo Engineering micrometer.
  • FIG. 31 shows the results of this experiment.
  • SleX given at 7 hours (both intranasally and intravenously) after the OVA challenge showed 70 - 74% reduction in footpad swelling relative to positive control mice as calculated using the formula set forth in Example 28.
  • SleX given intravenously or intranasally at 5 hours after footpad challenge showed 63% and 54% reduction in swelling respectively.
  • SleX given intervenously or intranasally at 10 hours after footpad challenge showed 58% and 32% reduction in swelling respectively.
  • mice Groups of Balb/c female mice were immunized with OVA 100 ⁇ g/mouse and DDA 20 ⁇ g /mouse in 100 ⁇ l of PBS intramuscularly into the hind leg muscle, while 100 ⁇ g /mouse of SleX was simultaneously administered intramuscularly, intranasally or intravenously. Seven day later the mice were footpad challenged with 20 ⁇ g /mouse of OVA in 20 ⁇ l of PBS. The footpad swelling was measured 24 hours later with a Mitutoyo Engineering micrometer.
  • FIG. 32 shows that administering SleX to the mice at the time of immunization produces the same level of suppression of the DTH inflammatory response regardless of the method by which the SleX is administered. This suggests that the SleX compound can be
  • LPS lipopolysaccharide caused lung injury is measured by weighing the lungs of sacrificed mice 24 hours after mice are given LPS intranasally. Briefly, groups of 8-10 week old Balb/c mice were sensitized with 5 ⁇ g /mouse of LPS in 50 ⁇ l of PBS intranasally under light anesthesia. Five hours later, 10 ⁇ g/mouse of SLeX, C19-9, T-disaccharide (#27) in 200 ⁇ l of PBS are given to the mouse
  • mice are sacrificed and the lungs removed and weighed.
  • SleA compound VII
  • SleX compound III
  • lymphocytes were cultured with varying concentrations of Mumps virus and varying concentrations of compound in RPMI 1640 medium (Gibco, Burlington, Ontario, Canada) supplemented with 5% Hybermix (Sigma, St. Louis, MO) at a density of 2 ⁇ 10 5 cells per well for three days at 37° C in 5% CO 2 . After three days the cells were pulsed for 6 hours with 1.0 ⁇ curies of 3 H -thymidine per well (Amersham Canada Ltd., Oakville, Ontario,
  • the cells were harvested using a PhD cell harvester and cpm determined in a Beckman 3000 scintillation counter. It has been shown previously that there is a direct correlation between the ability of lymphocytes to respond to an antigen and the amount of 3 H- thymidine incorporation 55 .
  • Lymphoproliferative responses are used in this example to measure T-helper cell responses in vitro.
  • FIG. 34 demonstrates that
  • SleX and SleA are able to inhibit an antigen specific lymphoproliferative response since the uptake of 3 H-thymidine is reduced relative to the untreated lymphocytes from mice exposed to the antigen. Without being limited to any theory, it is thought that SleX and SleA may interfere with macrophage T-cell interactions required in order to obtain a proliferative response in vitro.
  • Examples 36 and 37 illustrate the immunosuppressive properties of hexasaccharide glycoside 65a.
  • Example 36 Inhibition of DTH Inflammatory Response DTH inflammatory responses were measured using the mouse footpad swelling assay as described in Example 20. Briefly, groups of Balb/c mice were immunized with 10 ⁇ g of the L111 S-Layer protein. Seven days later, each group of mice was footpad-challenged with 10 ⁇ g of L-111 S-Layer protein. The resulting inflammatory footpad swelling was measured with a Mitutoyo Engineering micrometer 24 hours after challenge.
  • mice received 100 ⁇ g of this compound, injected into the tail vein, 5 hours after challenge.
  • Control groups received 100 ⁇ L of phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • hexasaccharide glycoside 65a had less than 50% of the footpad swelling as compared to the control mice.
  • mice treated with hexasaccharide glycoside 65a in Example 7 were re-challenged with L1 11 S-Layer protein 1 1 weeks after primary immunization.
  • mice treated with the PBS control responded with the usual degree of footpad swelling whereas mice treated with hexasaccharide glycoside 65a showed a reduction in footpad swelling of about 40%, i.e., the mice treated with hexasaccharide glycoside 65a exhibited only about 60% of the footpad swelling exhibited in mice treated with PBS.
  • hexasaccharide glycoside as per this invention also imparts tolerance to additional challenges from the same antigen.
  • hexasaccharide glycosides described herein in treating immune responses to an antigen and in inducing tolerance to the antigen in a mammal (mice).
  • a mammal mammal
  • such hexasaccharide glycosides will also be effective in treating human immune responses.
  • oligosaccharide glycosides related to blood group determinants could be used to suppress a cell-mediated immune response to an antigen by mere substitution for the oligosaccharide glycosides described in these examples.

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Abstract

Cette invention concerne des procédés et des compositions pharmaceutiques permettant de supprimer des réponses immunitaires à médiation cellulaire telles que les réponses inflammatoires à médiation cellulaire. Dans les compositions et les procédés décrits dans cette invention on utilise des glycosides d'oligosaccharide.
EP92911470A 1991-06-10 1992-06-09 Derives d'oligosaccharide immunosuppresseurs et tolerogenes Withdrawn EP0588852A1 (fr)

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US5559103A (en) * 1993-07-21 1996-09-24 Cytel Corporation Bivalent sialyl X saccharides
US5631283A (en) * 1994-02-02 1997-05-20 The United States Of America As Represented By The Secretary Of The Army Use of sialic acid or antibodies to sialidase as anti-infectious agents and anti-inflammatory agents
EP0727216B1 (fr) * 1994-07-15 2003-05-28 TAIYO KAGAKU Co., LTD. Composition medicamenteuse contenant un derive d'acide sialique
EP0926154B1 (fr) * 1996-07-23 2010-01-27 Seikagaku Corporation Nouveaux oligosaccharides a base de lactosamine et procede de preparation
CA2451051C (fr) * 2001-06-19 2012-11-27 Sorge Kelm Inhibiteurs de siglec
DE10204000A1 (de) * 2002-02-01 2003-08-14 Nutricia Nv Sialysierte Kohlenhydrate
EP1911850A1 (fr) * 2006-10-09 2008-04-16 Centre National De La Recherche Scientifique (Cnrs) Procédé de production de glycoprotéines et glycoconjugates 6-thio-sialylées
WO2009155108A1 (fr) 2008-05-30 2009-12-23 Momenta Pharmaceuticals, Inc. Structures saccharides et procédés destinés à fabriquer et à utiliser de telles structures
DK3116887T3 (da) 2014-03-13 2021-04-26 Univ Basel Carbonhydratligander, der binder til igm-antistoffer mod myelin-associeret glykoprotein
CN108026134A (zh) 2015-09-16 2018-05-11 巴塞尔大学 结合抗糖鞘脂糖蛋白表位抗体的碳水化合物配体

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EP0380084A3 (fr) * 1989-01-27 1991-03-13 The Biomembrane Institute Méthode d'inhibition des interactions cellule-cellule et cellule-substrat par blocage des interactions entre hydrates de carbone
EP0395217A3 (fr) * 1989-04-28 1991-01-23 The Biomembrane Institute Synthèse bio-organique de LeX (difucosyle y2; III3FucV3FucnLc6Cer) dimère et leurs analogues
US5109116A (en) * 1989-07-24 1992-04-28 Monsanto Company Immunosuppressive glycoprotein
EP0496832B1 (fr) * 1989-10-20 1996-12-11 New England Medical Center Inhibition de la liaison des cellules a mediation par padgem
AU8007791A (en) * 1990-06-15 1992-01-07 Cytel Corporation Intercellular adhesion mediators
US5211937A (en) * 1990-07-30 1993-05-18 Glycomed Incorporated Method of determining a site of inflammation utilizing elam-1 ligands
NZ240316A (en) * 1990-10-25 1996-12-20 Univ Michigan Compound for treating disease mediated by the elaboration of elam-1 on endothelial cells

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