+

WO1996009309A1 - Structure oligosaccharide d'un ligand de la selectine e et p - Google Patents

Structure oligosaccharide d'un ligand de la selectine e et p Download PDF

Info

Publication number
WO1996009309A1
WO1996009309A1 PCT/US1995/011401 US9511401W WO9609309A1 WO 1996009309 A1 WO1996009309 A1 WO 1996009309A1 US 9511401 W US9511401 W US 9511401W WO 9609309 A1 WO9609309 A1 WO 9609309A1
Authority
WO
WIPO (PCT)
Prior art keywords
ligand
essentially pure
types
selectin
oligosaccharides
Prior art date
Application number
PCT/US1995/011401
Other languages
English (en)
Inventor
Ake P. Elhammer
Jian-Guo Geng
Original Assignee
Pharmacia & Upjohn Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharmacia & Upjohn Company filed Critical Pharmacia & Upjohn Company
Priority to JP8510934A priority Critical patent/JPH10508580A/ja
Priority to AU35486/95A priority patent/AU3548695A/en
Priority to EP95932441A priority patent/EP0783508A1/fr
Publication of WO1996009309A1 publication Critical patent/WO1996009309A1/fr

Links

Classifications

    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • 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
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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

Definitions

  • This invention relates to the field of cell adhesion biology, E and P selectin and the oligosaccharide structure of a ligand for E and P selectin.
  • GlyCAM-1 a mucin-like molecule
  • the Le x determinant (Gal ⁇ l,4( ⁇ l,3Fuc)GlcNAc) is an important element of the P-selectin ligand on myeloid cells.
  • the ligand for P-selectin may be the same or very similar to that for E-selectin.
  • Tn (GalNAc) and T (Gal ⁇ l,3GalNAc), respectively, are known cancer antigens which appear in many carcinomas, e.g. colon and mammary carcinomas.
  • Tn (GalNAc) and T (Gal ⁇ l,3GalNAc) are known cancer antigens which appear in many carcinomas, e.g. colon and mammary carcinomas.
  • N-linked oligosaccharides Two common types of linkages for the attachment of oligosaccharide structures to glycoproteins are found in N-linked oligosaccharides, where the oligosaccharide structures are attached by an amide bond between the reducing N-acetylglucosamine on the oligosaccharide and an asparagine residue on the protein, and mucin type O-linked oligosaccharides which are conjugated via an ether bond between the reducing N-acetylgalactosamine on the oligosaccharide and a serine or threonine residue on the protein.
  • Other types of linkages between carbohydrate moieties and proteins do exist but these have little relevance for this discussion.
  • N-linked and mucin type O-linked oligosaccharides Several characteristics are shared by N-linked and mucin type O-linked oligosaccharides. Some similarities are: the majority of their component monosaccharides are the same; both types of structures, when synthesized by most untransformed mammalian cells, carries terminal sialic acids; both types of structures can carry blood group type substitutions such as Sialyl Lewis 1 (see below); and both types of oligosaccharides can contain poly-N-acetyllactosamine repeat structures. These observations, also suggested in the literature, indicate there may be a functional overlap between some N-linked and mucin type O-linked oligosaccharides. Fukuda, M. (1992) and Olden, K, Yeo, T.-K, and Yeo, -T.
  • Oligosaccharides are well positioned to act as recognition molecules due to their cell surface location and structural diversity. O-linked chains are of interest in view of the evidence that cell surface glycoproteins containing "clusters" of this type of substitution can form highly extended and rigid structures. Jentoft, N. (1990) Trends. Biochem. Sci. 15:291-294. Thus, these molecules are ideally positioned to perform recognition functions.
  • oligosaccharide structures can be created through the activities of a limited number of glycosyltransferases. Hence, these structures can be generated with relatively few gene products, suggesting a plausible mechanism for establishing the information necessary to direct a wide range of cell-cell interactions.
  • Examples of differential expression of cell surface carbohydrates and putative carbohydrate binding proteins, so called lectins, on interacting cells have been described Dodd, J., and Jessel, T.M., (1985) J. Neurosci., 5:3278; Regan, L.J., et al. (1986) Proc. Natl. Acad. Sci. USA, 83:2248; Constantine-Paton, M., et al. (1986) Nature, 324:159; and Tiemeyer, M., et al. (1989) J. Biol. Chem., 263:1671.
  • C-type lectins Animal lectins have been divided into three groups: C-type (Ca -dependent) lectins, S-type (soluble) lectins and P-type lectins. Drickamer, K. (1993) Annu. Rev. Cell Biol. 9:237-264.
  • An important group of C-type lectins are the selectins. The selectins have an important function in leucocyte extravasation at sites of inflammation.
  • Leukocytes are involved in the protection of the body against various microbial infections as well as diseases such as cancer.
  • white blood cells adhere to endothelial cells on the lumenal site of blood vessels, migrate to body tissues and accumulate there, they often cause damage, swelling, pain, and inflammation.
  • rheumatoid arthritis occurs when white blood cells enter the joints and attack the tissues.
  • cell adhesion receptors are called cell adhesion receptors.
  • the recruitment and extravasation of circulating leukocytes from the bloodstream across the endothelium into the target tissue, e.g. a site of injury or infection, are essential steps in the early response of inflammation. These processes are initiated by at least three families of cell surface adhesion molecules: the integrins, the immunoglobulin superfamily and the selectins. Springer, T.A. (1990) Nature 346:425; Hynes, R. O. and Lander, AD. (1992) Cell 68:303.
  • the selectins are a family of leukocyte-endothelial cell adhesion molecules, also referred to as LEC-CAM:s (Lectin EGF Complement regulatory-Cell Adhesion Molecule). Inflammation: Basic Principles and Clinical Correlates, Second Edition. J. I. Gallin et al, Eds, Raven Press, Ltd. NY 1992, pp 407-419. Selectin is the term favored by investigators working in the field. Bevilacqua M.P., et al. (1991) Cell 67:233. Selectins are generally believed to be primarily responsible for the initial interaction, called "rolling", of the leucocyte with the venular endothelium upon physiological and pathological stimuli, i.e.
  • leukocytes includes such cells as neutrophils, basophils, eosinophils, monocytes and T-cell subsets, de Bnujne-Admiraal, L.G. (1992) Blood 80:34.
  • L-selectin is a constitutively expressed lymphocyte homing receptor (a.k.a. peripheral lymph node homing receptor (pnHR), in a majority of leukocytes.
  • L- selectin is also known as: LECAM-1, LEC-CAM-1, LAM-1, gp90MEL, gplOOMEL, gpllOMEL, MEL-14, MEL- 14 antigen, Leu-8 (antigen), Ly-22 (antigen), TQ1 (antigen) and DREG.
  • E- selectin also known as ELAM-1, LECAM-2, and LEC-CAM-2
  • ELAM-1 a transient cytokine-inducible endothelial cell surface molecule which interacts with neutrophils, monocytes and memory T-cells.
  • P-selectin also known as LEC-CAM-3, LECAM-3, CD-62, GMP-140, and PADGEM, is rapidly expressed on the plasma membrane of endothelial cells and platelets during cellular activation and degranulation; it serves as a receptor for neutrophils and monocytes.
  • all selectins contain an NH 2 -terminal extracellular carbohydrate recognition domain (CRD) composed of 118 amino acid residues which is homologous to other C-type lectins, followed by a epidermal growth factor (EGF)- like domain, a variable number of complement regulatory protein-like repeats (different for different selectins), a transmembrane domain and a cytoplasmic tail.
  • CCD carbohydrate recognition domain
  • EGF epidermal growth factor
  • all selectins mediate Ca -dependent cell-cell contact by binding to oligosaccharide ligands on opposing cells.
  • selectins are known to interact with the oligosaccharide structures on other glycoproteins, called "selectin ligands," which are expressed on the surface of their opposing cells.
  • Immunoprecipitation experiments have shown that the construction of an L- ⁇ electin-IgG chimaera provides a compound that can be used to isolate selectin ligands.
  • a 50kD molecule and a 90kD molecule, both thought to be L-selectin ligands, has been specifically precipitated from mouse mesenteric lymph nodes using this type of compound. Imai, Y., et al. (1991) J. Cell Biol. 113:1213.
  • GlyCAM-1 mucin-like molecule
  • Recent work on the 90 kD ligand has revealed that the protein core of this molecule is identical to the vascular sialomucin CD34. Baumheuter, S., et al. (1993) Science 262:436.
  • a ligand has also been partially characterized for E-selectin.
  • a 150kD glycoprotein was isolated from a mouse neutrophil progenitor cell line using an E- selectin-IgG chimaera. Levinovitz, A., et al. (1993) J. Cell Biol. 121:449-459. See also Brandley et al., US 5,143,712, US 5,211,936, and US 5,211,937 patents.
  • a P- selectin ligand has similarly been identified.
  • a «120kD glycoprotein from human neutrophils has been identified in a 125 I-P-selectin blotting assay and by P-selectin affinity chromatography of H-glucosamine-labeled HL-60 cell extracts. Moore, K.L., et al. (1992) J. Cell. Biol. 118:445-456.
  • ligands for balh E- an ⁇ P-selectin have been described on mouse myeloid cells.
  • Two molecules, 150 kD and 160 kD which are monospecific ligands for E- and P-selectin, respectively, as well as two other molecules with molecular masses of 230 kD and 130 kD, respectively, that interact with both E- and P-selectin.
  • the present inventors report the structure of the O-linked oligosaccharides on a common ligand for E and P-selectin.
  • This molecule contains a heavy preponderance of O-linked oligosaccharides and fewer N-linked oligosaccharides.
  • the present inventors have found that the O-linked oligosaccharide structures on the ligand identified herein contains no Le x , Le a , sLe x or sLe a type substitutions.
  • the o-linked oligosaccharide structures on this ligand have little or no fucose.
  • the ligand described herein also contains little or no sulfated oligosaccharides.
  • the present inventors describe a truly unique ligand.
  • One object of the present invention is to provide a method for the purification of a selectin ligand. Another object of the invention is to provide a purified selectin ligand, specifically a common ligand for E and P-selectin. A further object of the present invention is to identify the 0-linked oligosaccharide structures on this ligand. Another aspect is to enable the preparation of glycosylation variants of selectin ligands, not otherwise found in nature. In another aspect, the invention provides a method of designing selectin inhibitors, mimicking carbohydrate based determinants of the selectin ligands.
  • the purified ligand, or fragments thereof, as well as carbohydrate and polypeptide components of the ligand, or antibodies to the ligand or to fragments thereof, can be used as inhibitors of binding of P- or E-selectin, or both, and in diagnostic assays and kits.
  • This invention encompasses those isolated molecules, fragments, combinations and variations thereof which adhere to E and or P selectin.
  • the isolated ligands can be generally characterized by the following embodiments, which may occur either separately or in various combinations and either in a free state or attached to a substrate.
  • 3GalNAc substituted with two sialic acids one sialic acid is at the 3 position of the terminal (non-reducing) Gal and one sialic acid is linked to the 6 position of the reducing GalNAc.
  • Figure 2. Separation of the O-linked oligosaccharides isolated from the HL-60 common ligand for E- and P-selectin on size -exclusion chromatography.
  • Figure 3. Size-exclusion separation of neutral and charged O-linked oligosaccharides isolated from the HL-60 common ligand for E- and P-selectin.
  • Figure 4. Sequential exoglycosidase digestion of an O-linked 12.8 GU oligosaccharide isolated from the HL-60 common ligand for E- and P-selectin.
  • Figure 7 Purification of the approximately 120 kD Ligand for P- and E- selectins. Additional Description of the Invention and the Preferred Embodiments Definitions, Abbreviations and Sources of Materials Some standard abbreviations used in connection with the present invention include: BSA, bovine serum albumin; DEAE, diethylaminoethyl; DMSO, dimethylsulfoxide; ELAM-1, endotheUal leukocyte adhesion molecule- 1; NANA, N-acetylneuraminic acid; TFA, trifluoroacetic acid; Tris, tris (hydroxymethyl) aminomethane.
  • BSA bovine serum albumin
  • DEAE diethylaminoethyl
  • DMSO dimethylsulfoxide
  • ELAM-1 endotheUal leukocyte adhesion molecule- 1
  • NANA N-acetylneuraminic acid
  • TFA trifluoroacetic acid
  • Tris tris (hydroxymethyl) aminomethane
  • C-type calcium-type
  • Gal galactose
  • GlcN glucosamine
  • GalN galactosamine
  • GlcNAc N-acetylglucosamine
  • GalNAc N-acetylgalactosamine
  • Fuc fucose
  • Gal-6S galactose 6-sulfate
  • Glc ⁇ Ac-6S N-acetylglucosamine 6-sulfate
  • GlcU-S glucuronic acid monosulfate
  • HEN high endotheUal venule
  • HPAEC high- pH anion-exchange chromatography
  • N-acetyUactosamine high-acetyUactosamine.
  • ⁇ eu ⁇ Ac N-acetylneuraminic acid
  • SA siaUc acid
  • sialyl Lewis x or sLe x ⁇ eu5Ac ⁇ 2 ⁇ 3Gal ⁇ l ⁇ 4(Fuc ⁇ l ⁇ 3)Glc ⁇ Ac
  • Lewis x or Le x Gal ⁇ l ⁇ 4(Fuc ⁇ l ⁇ 3)GlcNAc
  • Lewis a or Le a Gal ⁇ l ⁇ 3(Fuc ⁇ l ⁇ 4(GlcNAc
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
  • Ugand is used to describe any molecular structure of any size or shape which can bind to an appropriate receptor.
  • a naturally occuring Ugand for a selectin usually has a polypeptide backbone with oUgosaccharide sugars which interact with the selectin.
  • Ugand may refer to any structure which can act in the desired fashion and have the desired effect.
  • the Ugand may have only an oUgosaccharide portion.
  • the Ugand may have an oUgosaccharide portion with a backbone composed of natural or synthetic polypeptides, natural or synthetic polymers, nonpeptide organic compounds, inorganic compounds, substrates, or other suitable backbone materials.
  • a size limitation is not inherent in the term Ugand unless specified. Materials.
  • D-[6- H]Glucosamine hydrochloride 32 Ci/mmol was purchased from Amersham Corp., and D-[2- H(N)]mannose (21.0 Ci/mmol) is from New England Nuclear (NEN). ConcanavaUn A-Separose and QAE-Sephadex is from Pharmacia. Biogel P-6 is from BioRad. Jack bean ⁇ -galactosidase and Jack bean ⁇ -N- acetylglucosaminidase were purchased from Sigma. Xanthomonas manihotis ⁇ - galactosidase is from New England Biolabs. Arthrobacter ureafaciens neuraminidase is from Boehringer.
  • DMEM Dulbecco's- modified Eagle's Medium
  • FBS heat inactivated fetal bovine serum
  • trypsin penicillin and streptomycin
  • Gibco Gibco.
  • SuppUes are from standard sources unless otherwise indicated.
  • This activity has therapeutic value in the prevention or treatment of various blood related diseases and disorders.
  • the compounds may be used to inhibit platelet leukocyte rosetting and leukocyte adhesion to endotheUum.
  • leukocyte includes such ceUs as neutrophils, basophils, eosinophils, monocytes and T-cell subsets. See, de Bnnjne-Admiraal, L.G., Blood, vol. 80. pp. 134-142 (Aug.
  • the compounds are thus important to and may be used in the preparation of, making and using diagnostic assays and kits. Many procedures known in the art may be used to demonstrate inhibition of platelet leukocyte rosetting and leukocyte adhesion, thus producing a diagnostic kit or assay.
  • cardiovascular disorders including restenosis foUowing percutaneous transluminal coronary angioplasty (PTCA), myocardial ischemia, stroke, thrombo-emboUsm, deep vein thrombosis, acceleration of thrombolysis, lung injury associated with cardiopulmonary bypass; rheumatoid arthritis, asthma, pulmonary hypersensitivity diseases, organ transplantation, particularly kidney, heart and Uver organs; shock (septic and hemorrhagic), multiple organ injury syndromes; thermal injury; autoimmune disease, multiple sclerosis; inflammatory bowel diseases; infectious diseases including; general inflammation, angiogenesis and cancer metastasis, particularly breast, colon and lung metastasis and neuroblastoma.
  • PTCA percutaneous transluminal coronary angioplasty
  • myocardial ischemia stroke
  • thrombo-emboUsm deep vein thrombosis
  • acceleration of thrombolysis lung injury associated with cardiopulmonary bypass
  • rheumatoid arthritis asthma, pulmonary hypersensitivity diseases, organ transplantation, particularly kidney,
  • oUgosaccharides should not be considered in isolation, but in conjunction with the protein to which they are attached. See Rademacher et al, Ann. Rev. Biochem. 57, 785-838 (1988).
  • the novel agents of the present invention thus have various potential therapeutic uses of which the foUowing are iUustrative: Treatment of toxic and cachectic syndromes where cytokines (e.g. TNF, tumor necrosis factor ) are impUcated. e.g. maUgnancy, pre-eclamptic toxaemia, septic shock, Uver disease, respiratory distress syndrome. Chemotherapy of immunosuppressed states, e.g.
  • maUgnancy-associated immune-suppression iatrogenic immune-suppression (i.e. drugs).
  • Use of glycoprotein (EP) or multivalent (EPC) oUgosaccharides e.g. treatment of patients with graft vs host reactions (transplants), treatment of patients with host vs graft reactions (transplants) and treatment of maUgnant disease via activation of TNF.
  • the compounds of this invention may be used to coat heart valves and joint implants, particularly hip, knee and temporal-mandibular, to prevent leukocyte attachment. Coating of these devices with oUgosaccharides would also involve a polymer preparation in which the oUgosaccharides were incorporated into the polymer and then bonded to the device.
  • compositions comprising the carbohydrates, oUgosaccharides, proteins, Ugands, fragments thereof or their formulations are provided by the present invention.
  • the compounds of the invention such as various Ugands having the carbohydrate structure described by this invention or described by the claims of this invention can be administered to a subject in need thereof to treat the subject by either prophylactically preventing inflammation and/or reUeving it after it has begun.
  • Various deUvery systems are known and can be used for therapeutic delivery of the compounds.
  • the Ugands are preferably administered with a pharmaceutically acceptable carrier, the nature of the carrier differing with the mode of administration, for example, oral administration, usually using a soUd carrier and/or IN. administration of a Uquid solution carrier.
  • Methods of administration include but are not Umited to intravenous routes.
  • the amounts of oUgosaccharides administered to adult humans will be in the range of about 0.1 to 20 mg/kg, i.v. for 4-14 days. A more preferred range would be 0.5 to 2.5 mg kg doseage level.
  • the formulation of choice can be accompUshed using a variety of excipients including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin ceUulose, magnesium carbonate, and the like.
  • Oral compositions may be taken in the form of solutions, suspensions, tablets, piUs, capsules, sustained release formulations, or powders.
  • the subject Ugand molecules may be administered directly in transdermal formulations with permeation enhancers such as DMSO.
  • treating inflammation shall mean preventing or ameUorating inflammation and/or symptoms associated with inflammation.
  • compositions of the instant invention wiU contain from less than 1% to about 95% of the active ingredient, preferably about 10% to about 50%.
  • the frequency of administration wiU be determined by the care given based on patient responsiveness.
  • Other effective dosages can be readily determined by one of ordinary skill in the art through routine trials estabUshing dose response curves.
  • determining the dose of E or P selectin Ugands to be administered it must be kept in mind that one may not wish to completely block all of the E or P selectin receptors.
  • at least some of the white blood ceUs or neutrophils must be brought into the tissue in the areas where the wound, infection or disease state is occurring.
  • the amount of the E or P selectin Ugands administered as blocking agents must be adjusted carefully based on the particular needs of the patient while taking into consideration a variety of factors such as the type of disease that is being treated. It is beUeved that the Ugands or blocking agents of the present invention can be used to treat a wide range of diseases, including diseases such as rheumatoid arthritis and multiple sclerosis.
  • the compositions of the invention should be appUcable to treat any disease state wherein the immune system turns against the body causing the white cells to accumulate in the tissues to the extent that they cause tissue damage, swelling, inflammation and/or pain.
  • the inflammation of rheumatoid arthritis is created when large numbers of white blood ceUs quickly enter the joints in the area of disease and attack the surrounding tissues.
  • Formulations of the present invention might also be administered to prevent the undesirable after effects of tissue damage resulting from heart attacks.
  • a heart attack occurs and the patient has been revived, such as by the appUcation of anticoagulants or thrombolytic (e.g., tPA)
  • thrombolytic e.g., tPA
  • the endotheUal lining where a clot was formed has often suffered damage.
  • the antithrombotic has removed the clot, the damaged tissue beneath the clot and other damaged tissue in the endotheUal lining which has been deprived of oxygen become activated.
  • the activated endotheUal ceUs then express the E or P selectin within hours of the ceUs being damaged.
  • the selectin then extends into the blood vessels where they adhere to glycoprotein Ugand molecules on the surface of white blood ceUs.
  • Large numbers of white blood ceUs are quickly captured and brought into the tissue surrounding the area of activated endotheUal ceUs, resulting in inflammation, sweUing and necrosis which thereby decreases the likelihood of survival of the patient.
  • formulations of the invention In addition to treating patients suffering from the trauma resulting from heart attack, patients suffering from actual physical trauma could be treated with formulations of the invention in order to relieve the amount of inflammation and sweUing which no ⁇ naUy result after an area of the body is subjected to severe trauma.
  • Other disease states which might be treatable using formulations of the invention include various types of arthritis and adult respiratory distress syndrome.
  • the Ugand molecules of the invention can be formulated in suppositories and, in some cases, aerosol and intranasal compositions.
  • the vehicle composition will include traditional binders and carriers such as, polyalkylene glycols, or triglycerides.
  • Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciUary function.
  • Diluents such as water, aqueous saline or other known substances can be employed with the subject invention.
  • the nasal formulations may also contain preservatives such as, but not Umited to, chlorobutanol and benzalkonium chloride.
  • a surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.
  • the Ugand molecules of the instant invention may also be administered as injectables.
  • injectable compositions are prepared as Uquid solutions or suspensions; soUd forms suitable for solution in, or suspension in, Uquid vehicles prior to injection may also be prepared.
  • the preparation may also be emulsified or the active ingredient encapsulated in Uposome vehicles.
  • the Ugands in the form of glycoUpids and carbohydrates, or more specifically compounds described in CHART, 1 can be mixed with compatible, pharmaceutically acceptable excipients.
  • Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vehicle may contain minor amounts of auxiUary substances such as wetting or emulsifying agents or pH buffering agents.
  • auxiUary substances such as wetting or emulsifying agents or pH buffering agents.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack PubUshing Company, Easton, PA, 17th edition, 1985.
  • the composition or formulation to be administered will, in any event, contain a quantity of the Ugand molecules adequate to achieve the desired state in the subject being treated.
  • the various Ugand compounds of the present invention can be used by themselves or in combination with pharmaceutically acceptable excipient materials as described above. However, the Ugand compounds of the invention can be made as conjugates wherein the compounds of the invention are linked in some manner to a label.
  • the Ugand compounds of the invention act as biochemical delivery systems for the label so that a site of inflammation can be detected.
  • the Ugand molecules of the invention could also be used as laboratory probes to test for the presence of E or P selectin in a sample. Such probes are preferably labeled such as with a radioactive label.
  • Ligands in the form of glycoproteins having O-linked oUgosachharides which bind to E and P selectin are disclosed. More specificaUy, this invention relates to carbohydrate Ugands which bind to E and or P selectin and to compositions containing such Ugands which are useful in (a) determining the presence of E and or P selectin, (b) assaying for areas of inflammation, and (c) relieving inflammation by blocking the effects of E and or P selectin.
  • E and P selectin recognizes the oUgosaccharide structures on a mucin-like Ugand glycoprotein isolated from HL-60 ceUs in addition to a closely related protein, isolated from normal human leucocytes and developed the present invention.
  • Ligand molecules capable of binding to and interrupting the biological chain of events associated with E and or P selectin are disclosed.
  • the Ugand molecules act as biochemical blocking agents by binding to E and or P selectin and preventing circulating neutrophils from binding to stimulated endotheUal cells, thereby preventing a primary event of the inflammatory response.
  • the Ugands are in the form of O-Unked oUgosaccharides which can be labeled, bound to anti-inflammatory drugs and or formulated to provide: (1) compositions useful in assaying a sample for the presence of E and or P selectin, (2) compositions useful in detecting the site of inflammation in a patient, and (3) pharmaceutical composition useful in treating inflammation (or treating the inflammatory symptoms of certain diseases) or (4) blocking other effects involving the interaction of E and or P selectin and circulating neutrophils.
  • compositions which are useful in treating, preventing and/or alleviating any undesirable effects resulting from the interaction of E and or P selectin receptors and circulating neutrophils.
  • Such compositions are comprised of an inactive ingredient in the form of a pharmaceutically acceptable excipient material and a compound capable of binding to an E and or P selectin, the compounds having the general structural formula disclosed in CHART 1.
  • a primary object of the invention is to provide an E and or P selectin ligand in a useful formulation.
  • Another object is to provide a composition comprising an E and or P selectin Ugand which can be used to assay for the presence of E and or P selectin in a sample.
  • Another object is to provide a pharmaceutical formulation containing an E and or P selectin ligand which is useful in treating inflammation.
  • Ugands are in the form of non-toxic oUgosaccharides, and/or derivatives and mimetics thereof, which effectively bind the E and or P selectin and thereby block neutrophils from binding to the receptors in numbers which result in inflammation and/or other adverse effects.
  • a feature of the present invention is that the Ugand can be labeled and the labeled Ugand used in an assay to detect the presence of E and or P selectin in a sample.
  • Other features of the invention include the abiUty of pharmaceutical formulations of the invention to relieve the inflammatory symptoms of a wide range of diseases which are characterized by the binding of excessive amounts of leucocytes to a site.
  • a blood vessel waU is lined internally with endothelial cells.
  • the endotheUal ceUs can be activated, causing the ceUs to express E and or P selectin. Both red blood cells and white blood ceUs flow in the blood vessel.
  • the white blood ceUs display carbohydrate Ugands which have chemical and physical characteristics which aUow the Ugands to bind to the receptors, i.e. the selectins. Once the Ugand binds to the receptor, the white blood ceU is brought through the vessel wall into the surrounding tissue.
  • the white blood ceUs brought into the surrounding tissue can have positive effects, such as fighting infection, and negative effects, such as inflammation.
  • the present inventors have isolated and characterized Ugands apart from their presence on the surface of white blood ceUs. These isolated Ugands adhere to E and or P selectin by themselves and can be formulated into pharmaceutical compositions, which when administered will effectively block the E and or P selectin and prevent the adhesion of a receptor connected to a white blood ceU.
  • pharmaceuticaUy effective amounts of Ugands, some but not all, of the white blood ceUs wiU not reach the surrounding tissue.
  • inflammation can be prevented and or alleviated. It is known that for an acute inflammatory response to occur, circulating neutrophils must bind to and penetrate the vascular waU and access the site of injury.
  • the isolated Ugands can be generally characterized by the following embodiments, either separately or in various combinations:
  • CHART 1 The O-linked oUgosaccharide structures recovered from the common Ugand for E and P-selectin, isolated from HL-60 ceUs, are also shown and summarized in CHART 1.
  • CHART 1 summarizes five types of structures, named TYPES A-E, two additional types of structures not drawn in CHART 1 may also be present on the Ugand, they are referred to as TYPES F and G and are discussed in the section below labeled Obvious Variants and Preferred Compounds.
  • TYPES A-E two additional types of structures not drawn in CHART 1 may also be present on the Ugand, they are referred to as TYPES F and G and are discussed in the section below labeled Obvious Variants and Preferred Compounds.
  • CHART 1 contains two numbers separated by a slash, in a Unkage designation, it indicates that this Unkage has not been unequivocally determined; " ⁇ " at some terminal sialic acids indicates that this position may or may not be conjugated with
  • oUgosaccharides may be referred to by their corresponding size (without siaUc acids), in glucose units (GUs).
  • GU is a size designation.
  • a 12.8 GU oUgosaccharide does not necessarily contain any glucose, rather it would be the same size as a glucose oUgomer containing 12.8 units.
  • CHART 1 shows, from top to bottom, first, one type of oUgosaccharide, sized to 12.8 GU, that contains two sialic acid residues, as shown, this oUgosaccharide is named, TYPE A or 12.8 GU.
  • CHART 1 Second from the top in CHART 1 is an oUgosaccharide, sized to 9.8 GU, that may be in two forms, that is, it contains either one or two sialic acid residues at the locations shown, this oUgosaccharide is named, TYPE B or 9.8 GU. Third, is an oUgosaccharide, sized to 6.3 GU, that contains either zero, one (in either position) or two sialic acid residues, as shown, and is named, TYPE C or 6.3 GU. Fourth, is an oUgosaccharide, sized to 3.5 GU, that has either zero, one, or two sialic acid residue, named TYPE D or 3.5 GU.
  • the position of this substitution on the oUgosaccharide (on the terminal galactose or on the reducing N- acetylgalactosmine) has not been determined.
  • AU the oUgosaccharides except GalNAc by itself are core 1 based structures of comparatively simple design.
  • the 12.8, 9.8 and 6.3 GU oUgosaccharides are aU Unear, unbranched poly-N- acetyllactosamine repeat structures with the only variation between them being the number of N-acetyUactosamine repeat units and the sialylation pattern.
  • the o-linked oUgosaccharide structures on the HL-60 Ugand shares the N-acetyUactosamine feature previously reported for Ugands for E- and P-selectin but lacks Lewis type substitutions.
  • the m ⁇ ijority of the larger (12.8, 9.8 and 6.3 GU) structures are sialylated, again consistent with the reported requirement for sialic acid for selectin binding (Table 3).
  • Tn (GalNAc) and T (Gal ⁇ l,3GalNAc), respectively, are known cancer antigens which appear in many carcinomas, e.g colon and mammaiy carcinomas. See, Springer, G.F. (1984) Science 224:1198; Springer, G.F. (1989) Mol Immunol. 26:1; and Orntoft, T.F. et al. (1990) Int. J. Cancer 45:666.
  • CHART 1 may cooperatively generate a high affinity binding domain on the Ugand.
  • the Ugand described herein has features which may be described as mucin like: It is extensively O-glycosylated; there are three or fewer N-Unked oUgosaccharides on the Ugand.
  • the O-linked oUgosaccharides appear to form a tight cluster.
  • the molecule is largely resistant to degradation by pronase or trypsin.
  • neutrophils possess surface molecules which are referred to herein as Ugands and which Ugands have an affinity for and bind to receptor molecules on the surface of endotheUal ceUs which receptors are referred to herein as E or P selectin.
  • the larger molecules extracted which act as Ugands do so because they possess a particular molecular configuration at a particular point on the molecule required for adhesion to the E or P selectin.
  • the experiments herein were the first to isolate and characterize groups of molecules which do adhere to E or P selectin and thereafter specifically identify and characterize the oUgosaccharide structures of those molecules.
  • CHART 1 The general structural formula disclosed in CHART 1 encompasses a number of compounds which include characteristic features of isolated compounds which can adhere to E or P selectin receptors. Variations on the structures may also be possible provided those variations do not alter structural and or charge-related characteristics which would interfere with the adhesion of the Ugand to E or P selectin.
  • the present invention encompasses not only the molecules disclosed in CHART 1, and Types F and G discussed below, but various equivalents thereof which equivalents have an affinity for and an abiUty to adhere to E and or P selectin to the same or a greater degree than molecules of CHART 1 adhere to E and or P selectin.
  • the characteristics of such additional molecules can, of course, be confirmed using the adhesion assay described herein and/or related assays which test the adhesion of a putative Ugand to E and or P selectin under conditions of controlled detachment force.
  • each of the five or seven types of oUgosaccharides on the Ugand molecule are not intended to represent the total of the carbohydrates on the ligand.
  • the mucin like character of the Ugand suggests that it contains a large number of oUgosaccharide structures.
  • a similar Ugand molecule (which has been cloned) contains 53 predicted sites for o-glycosidic glycosylation. Sako et al, (1993) CeU 75:1179; Elhammer et al., (1993) J. Biol. Chem. 268:10029.
  • the selectins may be comprised of any combination of the different types of structures in any number.
  • the E or P selectin When the Ugand binds with the receptor, the E or P selectin, it binds using a selected combination of carbohydrates. Some combination is certainly needed, the range can be from one to seven types of oUgosaccharide(s), with five types, A-E, being preferred.
  • Varki concluded that this type of interaction between the selectins and their Ugands appear to be the most likely; he also described how the mucin like features of some of the Ugand molecules described to date easfly could accomodate this type binding "patch.”
  • Varki A(1994) Proc. Natl. Acad. Sci. USA 91:7390.
  • the ligand described herein has features which may be described as mucin like: It is extensively O-glycosylated; significant amounts of N-linked oUgosaccharides are also present on the molecule. The O-Unked oUgosaccharides appear to form a tight cluster and the molecule is largely resistant to degradation by pronase or trypsin.
  • Examples of specific oUgosaccharide Ugand structures are hereby provided.
  • Also described and claimed are the polyvalent forms where the oUgosaccharide combinations repeat themselves any number of times
  • possibiUties A, AA, AAA, B, BB, BBB, C, etc.
  • the order of the oUgosaccharides may be important.
  • One embodiment would aUow 3 of 7 possible oUgosaccharides in 3 unique possible combinations, repeated any number of times. This would aUow 7 x (7x7) or
  • possibilites 343 possibilites. Some of those possibiUties are shown here. AAA, AAB, AAC, AAD, AAE, AAF, AAG,
  • HL-60 ceUs were labeled with H-glucosamine (50 ⁇ Ci/ml) in normal RPMI medium for 48 hours. Preparation of the P-selectin ligand.
  • a P-selectin affinity column (10 mg of purified human platelet P-selectin coupled onto 10 ml of Affi-Gel 15 resins) and a bovine serum albumin guard column were prepared as described by Ma et al, (1994) J. Biol. Chem. 269, 27739-27746.
  • the labeled ceUs were lysed with detergent as described by Moore et al. ((1992) J. CeU Biol. 118, 445-456) and the ceU lysates were appUed to the P-selectin affinity column through a BSA guard column in the presence of CaCl 2 .
  • Glycopeptides were prepared from the intact purified radiolabeled ligand molecule by digestion with Pronase (2 mg ml) in 100 mM Tris-HCl, pH 8.0, 20 mM CaCl 2 at 55°C for 4 hours.
  • FIGURE 7 Purification of -120 kD Ligand for P- and E-selectins HL-60 ceUs labeled with [3H]GlcNAc were lysed with Triton X-100 and separatedly loaded onto P-selectin affinity column or E-selectin receptor-globuUn affinity column in the presence of Ca . After extensive washing, the bound materials were eluated with EDTA and were subject to 7% SDS-PAGE (under reducing conditions) foUowed by autoradiography. Lane 1, the eluate from P-selectin affinity column; lane 2, eluates from E-selectin receptor-globuUn affinity column. Enzyme Treatments.
  • Digestion with Arthrobacter ureafaciens neuraminidase (2 U/ml) was done in 100 mM sodium acetate buffer, pH 5.0 for 18 hours; jack bean ⁇ -galactosidase (400mU/ml) was in 50 mM citrate-phosphate, pH 4.6, for 48 hours; jack bean ⁇ -N- acetylglucosaminidase (4.15 mU/ml) was in 50 mM sodium citrate, pH 5.6, for 18 hours; Xanthomonas manihotis ⁇ -galactosidase (167 U/ml) was in 50 mM sodium citrate, pH 4.5, for 18 hours; almond ⁇ -fucosidase (0.2 mU/ml) was in 50 mM sodium citrate, pH 5.0, for 18 hours. AU enzyme digestions were carried out at 37° C under toluene atmosphere.
  • Glycopeptides prepared by digestion of the intact [ 3 H]-glucosamine labeled Ugand molecule with pronase, were separated on ConcanavaUn A-Sepharose. This resulted in essentially all of the appUed radioactivity eluting in the break-through fractions from the column, insignificant amounts of radioactivity was eluted by the hapten sugars, ⁇ -methylmannoside and ⁇ -methylglucoside, suggesting that the Ugand molecule does not contain any high-mannose, hybrid or bi-antennary complex type oUgosaccharides.
  • the total O-Unked oUgosaccharides were cleaved from the purified [ H]- glucosamine labeled Ugand molecule by mild alkaline sodium borohydride treatment.
  • the released oUgosaccharides were then fractionated on QAE-Sephadex and charged structures were eluted with step-wise increases NaCl concentration ( Figure 1 and Table 1).
  • FIGURE 1 Separation of labeled O-Unked oUgosaccharides isolated from the HL-60 common Ugand for E- and P-selectin, on QAE-Sephadex. Charge separation of the O-linked oligosaccharide structures.
  • Re-chromatography of the charged fractions after digestion with Arthrobacter ureafaciens neuraminindase, resulted in the majority of the radioactivity in both charged fractions eluting in the QAE-Sephadex column break-through fractions, and the remaining portion (24% and 25% for the 70 mM and 20 mM eluates, respectively) eluted in the fractions containing material with one charge.
  • the radioactive material in these neuraminidase resistant, charged fractions was identified by paper chromatography.
  • FIGURE 2 shows separation of the complete mixture of O-Unked oUgosaccharides on the molecule.
  • the mixture of sugars separated into five peaks eluting in the positions of oligomers composed of 12.8, 9.8, 6.3, 3.5, and 2.5 glucose units (GU), respectively.
  • TABLE 2 shows the distribution of radioactivity in the five peaks on the chromatogram.
  • FIGURE 3 shows a size separation of the portions of the structures containing none, one and two charges, respectively. See, TABLE 3, FIGURE 2, and FIGURE 3.
  • FIGURE 2 Separation of the O-Unked oUgosaccharides isolated from the HL-60 common Ugand for E- and P-selectin on size -exclusion chromatography.
  • Total O-Unked oUgosaccharides were isolated from the purified in vivo [ H]- glucosamine labeled E/P-selectin Ugand, siaUc acids were removed by neuraminidase digestion and the resulting neutral oUgosaccharides were separated on a GlycoMap column.
  • the five radioactive peaks (from right to left) on the chromatogram have elution volumes corresponding to glucose oUgomers composed of 12.8, 9.8, 6.3. 3.5 and 2.5 units respectively.
  • FIGURE 3 Size-exclusion separation of neutral and charged O-Unked oUgosaccharides isolated from the HL-60 common Ugand for E- and P-selectin.
  • the total [ H]-glucosamine labeled O-Unked oUgosaccharides from the H -60 Ugand were fractionated on QAE-Sephadex as described in FIGURE 1.
  • FoUowing removal of sialic acid from the charged oUgosaccharides by neuraminidase digestion, neutral oUgosaccharides (Panel A) and oUgosaccharides containing one (Panel B) and two siaUc acids (Panel C) were fractionated separately on the GlycoMap column. Only the portions of the chromatograms containing measurable amounts of radioactivity are shown in the figure.
  • FIGURE 4 shows the sequential degradation of the largest (12.8 GU) oUgosaccharide. Initially, this structure was digested sequentially with jack bean ⁇ - galactosidase and jack bean ⁇ -N-acetylglucosaminidase; this resulted in a one and two GU shift in elution volume, respectively.
  • a 2 GU radioactive digestion product was released by the ⁇ -N-acetylglucosaminidase digestion. These are the predicted results for the removal of one terminal N-acetyUactosamine unit (FIGURE 4, top panel). The released radioactivity was identified as N-acetylglucosamine by paper chromatography. The product from these two digestions eluted at 9.8 GU (FIGURE 4, top panel). As is apparent from FIGURE 4, the first ⁇ -N-actylglucosaminidase digestion of the 12.8 GU oUgosaccharide did not result in degradation of all the radiactive material in this peak; some radioactivity also eluted at 11.8 GU after digestion.
  • FIGURE 4 Sequential exoglycosidase digestion of an O-Unked 12.8 GU oUgosaccharide isolated from the HL-60 common Ugand for E- and P-selectin.
  • the 12.8 GU oUgosaccharide isolated from fractionation of the total O-Unked oUgosaccharides on the HL-60 Ugand was subjected to sequential exoglycosidase digestion and re-chromatography on the GlycoMap column as described in the Experimental Procedures.
  • the elution positions of the glucose oUgomer standards are indicated by the arrows at the top of the chromatograms; the numbers identifies the size (in GU) of the individual standard molecules.
  • the key identifies elution profiles obtained for the undigested molecule and for products from digestions with jack bean ⁇ -N-acetylglucosaminidase and jack bean ⁇ -galactosidase, respectively.
  • E elution volume (ml); R, radioactivity.
  • the two GU fragments released by the ⁇ -N-acetylglucosaminidase digestions and the final 3.5 GU degradation product were identified as N-acetylglucosamine and Gal ⁇ l,3GalNAc-ol, respectively, on paper chromatography (FIGURE 5, panels A and B; the chromatography standards are: 1, Gal ⁇ l,3GalNAc-ol and 2, GalNAc-ol).
  • FIGURE 5 Separation of radioactive fragments obtained from exoglycosidase digestion of the 12.8 GU O-linked oUgosaccharide on the HL-60 common Ugand for E- and P-selectin.
  • the 2 and 3.5 GU fragments generated by exoglycosidase digestion of the 12.8 GU oUgosaccharide were separated on descending paper chromatography in pyridine-ethyl acetate-glacial acetic acid-water (5:5:1:3).
  • Panels A and B shows separations of the 2 and 3.5 GU exoglycosidase digestion fragments, respectively.
  • the standards were 1, Gal ⁇ l,3GalNAc-ol; 2, GalNAc-ol.
  • Digestion of the 6.3 GU oUgosaccharide with jack bean ⁇ -galactosidase resulted in a one GU shift in elution volume on the column, indicating the removal of one terminal galactose.
  • Digestion of the 6.3 GU oUgosaccharide with Xanthomonas manihotis ⁇ -galactosidase did not result in a shift in elution volume indicating that the terminal galactose on this structure is linked 1,4 to N- acetylglucosamine.
  • the two smaUer O-Unked structures on the Ugand molecule were identified on paper chromatography.
  • the 3.5 GU oUgosaccharide co-migrated with the Gal ⁇ l, 3GalNAc-ol standard and digestion of this structure with jack bean and bovine testis ⁇ -galactosidase followed by re-chromatography on paper, revealed its identity with this standard.
  • FIGURE 6 Identification of the 3.5 and 2.5 GU O-Unked oUgosaccharides on the HL-60 common Ugand for E- and P-selectin.
  • a Numbers in parenthesis represents percent of total identified structures.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Pulmonology (AREA)
  • Immunology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Endocrinology (AREA)
  • Reproductive Health (AREA)
  • Diabetes (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Saccharide Compounds (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

On a isolé à partir des cellules HL-60 marquées in vivo par [3H] glucosamine un ligand spécifique de la sélectine E et P par chromatographie combinée de l'agarose agglutinine de germe de blé et de la rIg-agarose de la sélectine E ou P. Les oligosaccharides totaux à liaison de type O sur la molécule purifiée sont libérés par hydrolyse alcaline en présence de NaBH et analysés par chromatographie par échange d'ions, par tamisage moléculaire et sur papier en combinaison avec des digestions spécifiques à l'aide de l'exoglycosidase. On décrit divers ligands et glucides dérivés.
PCT/US1995/011401 1994-09-20 1995-09-15 Structure oligosaccharide d'un ligand de la selectine e et p WO1996009309A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP8510934A JPH10508580A (ja) 1994-09-20 1995-09-15 Ｅおよびｐセレクチンに対するリガンドのオリゴ糖構造
AU35486/95A AU3548695A (en) 1994-09-20 1995-09-15 The oligosaccharide structure of a ligand for e and p selectin
EP95932441A EP0783508A1 (fr) 1994-09-20 1995-09-15 Structure oligosaccharide d'un ligand de la selectine e et p

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US30901294A 1994-09-20 1994-09-20
US43329895A 1995-05-03 1995-05-03
US08/433,298 1995-05-03
US08/309,012 1995-05-03

Publications (1)

Publication Number Publication Date
WO1996009309A1 true WO1996009309A1 (fr) 1996-03-28

Family

ID=26976573

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/011401 WO1996009309A1 (fr) 1994-09-20 1995-09-15 Structure oligosaccharide d'un ligand de la selectine e et p

Country Status (4)

Country Link
EP (1) EP0783508A1 (fr)
JP (1) JPH10508580A (fr)
AU (1) AU3548695A (fr)
WO (1) WO1996009309A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999043834A3 (fr) * 1998-02-27 1999-11-18 Genetics Inst Proteine constituant un ligand de la p-selectine et proteines de fusion tetrameriques
EP0847766A3 (fr) * 1996-12-10 2000-08-30 SORIN BIOMEDICA S.p.A. Dispositif pour implantation et trousse contenant ce dispositif
US6258796B1 (en) 1996-11-20 2001-07-10 The University Of Montana Water soluble lipidated arabinogalactan
WO2002089819A1 (fr) * 2001-05-07 2002-11-14 Vereniging Voor Christelijk Wetenschappelijk Onderwijs Glycoconjugues et utilisations de ceux-ci
WO2004033473A1 (fr) 2002-10-11 2004-04-22 Yamanouchi Europe B.V. Composes a base de glucose presentant une affinite pour la selectine-p

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991019501A1 (fr) * 1990-06-15 1991-12-26 Cytel Corporation Mediateurs d'adherence intercellulaire
WO1992022563A1 (fr) * 1991-06-10 1992-12-23 Alberta Research Council Composes modifies de lewisa au sialyl
WO1994026759A1 (fr) * 1993-05-14 1994-11-24 The Regents Of The University Of California Compositions de modulation du recepteur de selectine
WO1994026760A1 (fr) * 1993-05-14 1994-11-24 Cytel Corporation ANALOGUES DE SIALYLE Lex UTILES EN TANT QU'INHIBITEURS DE L'ADHESION CELLULAIRE

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991019501A1 (fr) * 1990-06-15 1991-12-26 Cytel Corporation Mediateurs d'adherence intercellulaire
WO1992022563A1 (fr) * 1991-06-10 1992-12-23 Alberta Research Council Composes modifies de lewisa au sialyl
WO1994026759A1 (fr) * 1993-05-14 1994-11-24 The Regents Of The University Of California Compositions de modulation du recepteur de selectine
WO1994026760A1 (fr) * 1993-05-14 1994-11-24 Cytel Corporation ANALOGUES DE SIALYLE Lex UTILES EN TANT QU'INHIBITEURS DE L'ADHESION CELLULAIRE

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6258796B1 (en) 1996-11-20 2001-07-10 The University Of Montana Water soluble lipidated arabinogalactan
US6303584B1 (en) 1996-11-20 2001-10-16 The University Of Montana Water soluble lipidated arabinogalactan
EP0847766A3 (fr) * 1996-12-10 2000-08-30 SORIN BIOMEDICA S.p.A. Dispositif pour implantation et trousse contenant ce dispositif
US6251142B1 (en) 1996-12-10 2001-06-26 Sorin Biomedica Cardio S.P.A. Implantation device and a kit including the device
WO1999043834A3 (fr) * 1998-02-27 1999-11-18 Genetics Inst Proteine constituant un ligand de la p-selectine et proteines de fusion tetrameriques
WO2002089819A1 (fr) * 2001-05-07 2002-11-14 Vereniging Voor Christelijk Wetenschappelijk Onderwijs Glycoconjugues et utilisations de ceux-ci
WO2004033473A1 (fr) 2002-10-11 2004-04-22 Yamanouchi Europe B.V. Composes a base de glucose presentant une affinite pour la selectine-p

Also Published As

Publication number Publication date
AU3548695A (en) 1996-04-09
EP0783508A1 (fr) 1997-07-16
JPH10508580A (ja) 1998-08-25

Similar Documents

Publication Publication Date Title
US5143712A (en) Method of determining a site of inflammation utilizing elam-1 ligands
AU657275B2 (en) New carbohydrate-based anti-inflammatory agents
Sabesan et al. Combined chemical and enzymatic synthesis of sialyloligosaccharides and characterization by 500-MHz and proton and carbon-13 NMR spectroscopy
Tyrrell et al. Structural requirements for the carbohydrate ligand of E-selectin.
Sarkar et al. Fucosylation of disaccharide precursors of sialyl LewisX inhibit selectin-mediated cell adhesion
AU665431B2 (en) New carbohydrate-based anti-inflammatory agents
Yamashita et al. Urinary glycopeptides of fucosidosis.
KRUSIUS et al. Characterization of a novel sugar sequence from rat‐brain glycoproteins containing fucose and sialic acid
US5211936A (en) Method of determining a cite of inflammation utilizing elam-1 ligands site
Seppo et al. Synthesis of a tetravalent sialyl Lewis x glycan, a high-affinity inhibitor of L-selectin-mediated lymphocyte binding to endothelium
Aeed et al. Characterization of the O-linked oligosaccharide structures on P-selectin glycoprotein ligand-1 (PSGL-1)
Chandrasekaran et al. Sialoglycoproteins of human mammary cells: partial characterization of sialoglycopeptides
AU678373B2 (en) Substituted lactose derivatives as cell adhesion inhibitors
AU713078B2 (en) Synthetic multivalent sLEX containing polylactosamines and methods for use
Adamany et al. A carbohydrate structural variant of MM glycoprotein (glycophorin A).
EP0783508A1 (fr) Structure oligosaccharide d'un ligand de la selectine e et p
Fukuda et al. Isolation and characterization of alkali-labile oligosaccharide units from horse glycophorin
Marshall et al. Interaction between complement proteins C5b-7 and erythrocyte membrane sialic acid.
Tamura Recent advances in the synthetic studies of glycosaminoglycans
Natunen et al. Enzymatic synthesis of two lacto-N-neohexaose-related Lewis x heptasaccharides and their separation by chromatography on
Kawashima et al. Isolation and characterization of alkali-labile oligosaccharide units from porcine erythrocyte glycophorin
WO1999012944A2 (fr) POLYLACTOSAMINES SYNTHETIQUES CONTENANT UN sLex DIVALENT ET SES METHODES D'UTILISATION
Edge et al. Structure of the O-linked oligosaccharides from a major thyroid cell surface glycoprotein
EP0830365B1 (fr) Analogues kojibiosides modifies
US20030186935A1 (en) Myeloglycan

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TT UA US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref country code: US

Ref document number: 1997 809258

Date of ref document: 19970313

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1995932441

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1995932441

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1995932441

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载