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  • 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
  • C07H15/10—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical containing unsaturated carbon-to-carbon bonds
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  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
• • A—HUMAN NECESSITIES
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  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
  • A61K38/13—Cyclosporins
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  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
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  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
  • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
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  • 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
• • 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
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1048—Glycosyltransferases (2.4)
  • C12N9/1051—Hexosyltransferases (2.4.1)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56966—Animal cells
  • G01N33/56972—White blood cells

Definitions

• the present invention relates to compositions and methods for reducing or controlling inflammation and for treating inflammatory disease processes and other pathological conditions mediated by intercellular adhesion.
• Vascular endothelial cells and blood platelets play key roles in a number of biological responses by selectively binding certain cells, for instance phagocytic leukocytes, in the blood stream.
• endothelial cells for instance phagocytic leukocytes
• Certain inflammationtriggering compounds are known to act directly on the vascular endothelium to promote the adhesion of leukocytes to vessel walls, which cells then move through the walls and into areas of injury or infection.
• Cellular adhesion to vascular endothelium to promote the adhesion of leukocytes to vessel walls, which cells then move through the walls and into areas of injury or infection.
• endothelium is also thought to be involved in tumor metastasis. Circulating cancer cells apparently take advantage of the body's normal inflammatory mechanisms and bind to areas of blood vessel walls where the endothelium is activated.
• Platelets are also involved in similar responses. Platelets are known to become activated during the initiation of hemostasis and undergo major morphological, biochemical, and functional changes (e.g., rapid granule exocytosis, or degranulation), in which the platelet alpha granule membrane becomes fused with the external plasma
• Activated platelets are recruited into growing thrombi or are cleared rapidly from the blood circulation. Activated platelets are known to bind to phagocytic leukocytes, including monocytes and neutrophils. Examples of pathological and other biological processes which are thought to be mediated by this process include atherosclerosis, blood clotting and inflammation.
• ELAM-1 endothelial leukocyte adhesion molecule-1
• GMP-140 granule membrane protein-140
• ELAM-1 has been shown to mediate endothelial leukocyte adhesion, which is the first step in many
• ELAM-1 binds human neutrophils, monocytes, eosinophils, certain T-lymphocytes (N. Graber et al., J. Immunol., 145:819 (1990)), NK cells, and the promyelocytic cell line HL-60.
• selectin has been suggested for a general class of receptors, which includes ELAM-1 and GMP-140, because of their lectin-like domain and the selective nature of their adhesive functions. These cell surface receptors are expressed on a variety of cells. GMP-140 (also known as PADGEM) is present on the surface of platelets and endothelial cells, where it mediates platelet-leukocyte and endothelium-leukocyte interactions. Another member of the selectin class is the MEL-14 antigen and its human analog LAM-1 which are cell surface receptors of lymphocytes, and act as lymph node homing
• the ligand(s) could also be used to target other pharmaceutical compounds, such as anti-inflammatory agents or anti-oxidants, to the sites of injury. To date, however, insufficient understanding of the interaction of the ligand(s) and receptor molecules on the respective cells has hindered these efforts.
• the present invention fulfills these and other related needs.
• Novel compositions which selectively bind a selectin cell surface receptor and which have at least one
• compositions inhibit intercellular adhesion mediated by the selectin cell surface receptor and thereby are capable, for example, of inhibiting inflammatory and other pathological responses associated with cellular adhesion.
• the composition comprises sialic acid and fucose, a sulfate, or a phosphate.
• the composition that binds the selectin may be a glycoprotein, a glycolipid, or an
• compositions are provided.
• the pharmaceutical compositions can be, for example, liposomes which comprise a ligand oligosaccharide moiety capable of selectively binding a selectin receptor and a pharmaceutically acceptable carrier.
• the liposome containing the ligand may also serve as a targeting vehicle for a
• chemotherapeutic agent which agent is contained within the liposome and delivered to targeted cells which express a selectin receptor.
• the chemotherapeutic agent is an anti-inflammatory agent or an anti-oxidant.
• liposomes encapsulated within liposomes is a convenient and effective method for reducing therapeutic levels of a drug and minimizing side effects.
• the invention comprises methods of inhibiting intercellular adhesion in a patient for a disease process such as inflammation or reperfusion injury by
• a therapeutically effective dose of a compound comprising a moiety capable of binding a selectin cell surface receptor may be administered to the patient a therapeutically effective dose of a compound comprising a moiety capable of binding a selectin cell surface receptor.
• the cell surface receptor such as ELAM-1 or GMP-140, may be expressed on vascular endothelial cells or platelets.
• the inflammatory process may be, for example, rheumatoid arthritis.
• administered may have an oligosaccharide moiety having the chemical formula: NeuAc ⁇ 2, 3Gal ⁇ 1,4 (Fuc ⁇ l, 3) GlcNAc ⁇ 1-R 1 ;
• R 1 is an amino acid, oligopeptide, lipid, or
• Fig. 1 illustrates the ability of cells which express SLX (LEC 11) to bind to IL-1 ⁇ activated endothelial cells compared to those cells which express non-sialylated Le x (CHO-K1 and LEC 12).
• Fig. 2 illustrates the ability of monoclonal antibodies specific for SLX to block selectin-mediated binding of HL-60 cells at 37oC (Fig. 2A) and 4oC (Fig. 2B) compared to monoclonal antibodies which do not bind SLX determinants.
• Fig. 3 illustrates the effects of incubating LEC 11 (Fig. 3A) and LEC 12 (Fig. 3B) cells with SLX and non-SLX specific monoclonal antibodies on binding to activated
• Fig. 4 illustrates the results obtained by treating
• HL-60, LECH and LEC12 cells with sialidase before binding to activated endothelial cells.
• Fig. 5 compares the ability of liposomes which contain glycolipids with SLX, Le x , or similar carbohydrate structures to inhibit the binding of HL-60 cells to activated endothelial cells.
• Fig. 6 compares the inhibition of GMP-140 mediated platelet adhesion by monoclonal antibodies specific for SLX and Le x determinants.
• Fig. 7 compares the ability of liposomes which contain glycolipids with SLX, Le x , or similar carbohydrate structures to inhibit the binding of HL-60 cells to activated platelets.
• Fig. 8 compares the ability of liposomes which contain glycolipids with SLX, Le x , or similar carbohydrate structures to inhibit the binding of PMNs to activated
• Fig. 9 shows inhibition of GMP-140 mediated adehsion by glycolipids with the terminal sialic acid either NeuAc or NeuGc.
• compositions and methods are provided for inhibiting inflammatory and other disease responses mediated by cellular adhesion.
• the present invention also provides compounds (e.g., glycoconjugates and monoclonal antibodies) which have the ability to block or inhibit the adhesion of the cells mediated by selectin cell surface receptors. Methods for preparing and screening for such compounds are also provided. In addition, diagnostic and therapeutic uses for the compounds are provided.
• selectin cell surface receptors As discussed above, selectins, also known as the "LEC-CAM” family of cell adhesion molecules, are unique
• glycoproteins expressed on the surface of a variety of cells For instance, ELAM-1 is inducibly expressed on vascular endothelial cells.
• IL-I ⁇ interleukin I ⁇
• TNF ⁇ tumor necrosis factor ⁇
• bacterial endotoxin lipopolysaccharide
• GMP-140 is a membrane
• glycoprotein of platelet and endothelial secretory granules glycoprotein of platelet and endothelial secretory granules
• Activated platelets which express GMP-140 on their surface are known to bind to monocytes and
• GMP-140 is an alpha granule membrane protein of molecular weight 140,000 that is expressed on the surface of activated platelets upon platelet stimulation and granule secretion (Hsu-Lin et al., J. Biol. Chem. 259:9121-9126 (1984); Stenberg et al., J. Cell Biol. 101:880-886 (1985); Berman et al., J. Clin. Invest.
• a third selectin receptor is the lymphocyte homing receptor, MEL-14 antigen or LAM-1 (Gallatin et al., Nature
• MEL-14 antigen/LAM-1 is believed to function early in neutrophil binding to the endothelium.
• selectin receptors The structure and function of selectin receptors has been elucidated by cloning and expression of full length cDNA encoding each of the above receptors (see, e.g., Bevilacqua et al., Science, supra, (ELAM-1), Geng et al., supra. (GMP 140), and Lasky et al., supra. (MEL-14 antigen)).
• the extracellular portion of selectins can be divided into three segments based on homologies to previously described proteins.
• the N-terminal region (about 120 amino acids) is related to the C-type
• mammalian lectin protein family as described by Drickamer, J. Biol. Chem.. 263: 9557-9560 (1988) (which is incorporated herein by reference) that includes low affinity IgE receptor CD23.
• a polypeptide segment follows, which has a sequence that is related to proteins containing the epidermal growth factor (EGF) motif.
• EGF epidermal growth factor
• tandem repetitive motifs of about 60 amino acids each, related to those found in a family of complement regulatory proteins.
• selectin receptors comprise a lectin-like domain
• the specificity of the molecules is likely to be based on protein-carbohydrate interactions.
• Evidence provided here indicates that a sialylated, fucosylated N-acetyllactosamine unit of the Lewis X antigen, designated here as SLX, is a moiety recognized by the lectin region of the selectin
• Compounds of the present invention comprise this fucosylated, sialylated N- acetyllactosamine unit in a variety of configurations.
• 2-N-acetylglucosamine is represented by GlcNAc
• fucose is Fuc
• galactose is Gal
• glucose is Glc.
• Two sialic acids which may be present on the oligosaccharides of the present invention are 5-N-acetylneuraminic acid (NeuAc) and 5-N-glycolylneuraminic acid (NeuGc).
• NeuAc 5-N-acetylneuraminic acid
• NeuGc 5-N-glycolylneuraminic acid
• all sugars except fucose (L-isomer) are D-isomers in the cyclic pyranose configuration.
• the two anomers of the cyclic forms are represented by ⁇ and ⁇ .
• the monosaccharides are generally linked by glycosidic bonds to form oligo- and polysaccharides.
• the orientation of the bond with respect to the plane of the rings is indicated by ⁇ and ⁇ .
• the particular carbon atoms that form the bond between the two monosaccharides are also noted.
• a ⁇ glycosidic bond between C-1 of galactose and C-4 of glucose is represented by Gal ⁇ 1, 4Glc.
• D-sugars e.g., D- GlcNAc, D-Gal, and D-NeuAc
• hydroxyl attached to C-1 (C-2 in NeuAc). is below the plane of the ring and ⁇ is above the ring.
• the ⁇ designation means the hydroxyl is above the ring and ⁇ means it is below.
• SLX as a carbohydrate ligand that mediates leukocyte-endothelial and leukocyte-platelet cell adhesion
• compounds comprising SLX or its mimetics can be purified or synthesized de novo. Once obtained, such compounds can be used for a variety of purposes, including, for example, competitive inhibition of the binding of SLX-bearing cells to cells which express the selectin receptors.
• compounds which contain one or more SLX-R units or mimetics can serve as effective inhibitors of, for instance, inflammation, atherosclerosis, clotting and other endothelial or platelet-mediated pathologies.
• SLX antigen is present on N-linked carbohydrate groups of the cell surface glycoproteins of LEC11 cells, a glycosylation mutant of CHO cells.
• LEC11 expresses this unique glycopeptide which contains a terminal structure bearing both sialic acid and fucose in the SLX sequence:
• the sialic acid is in the form of NeuAc.
• the sialic acid may be in other forms, such as NeuGc, without significantly affecting binding.
• SLX isolated from bovine erythrocytes comprises NeuGc.
• the affinity for selectin receptors is the same for both forms.
• SLX refers to the minmal tetrasaccharide unit shown above in which the terminal sialic acid is NeuAc, NeuGc or other equivalent forms of sialic acid. Structures illustrated herein which show the sialic acid residue as NeuAc are understood to include these other forms, in particular NeuGc.
• SY2 also known as the VIM antigen
• oligosaccharides recognized by selectin receptors may comprise a number of the sialylated N-acetyllactosamine units, at least one of which is fucosylated (see, Teimeyer et al., Proc. Natl. Acad. Sci. (USA) 88:1138-1142 (1991), which is incorporated herein by reference.
• Sources that can be used to obtain the SLX unit include any cell which naturally expresses the moiety on glycolipid or glycoprotein carbohydrate groups.
• polymorphonuclear neutrophils have been used to purify this unit.
• Other cells which bind to activated vascular endothelium can also be used to isolate the ligand (see. Symington et al., J. Immunol.
• LEC11 cells can be used to obtain glycoprotein or glycolipid which contains the SLX unit using, for instance, the method described in Stanley et al., supra. Briefly, in one method LEC11 cells are infected with vesicular stomatitis virus. The structural carbohydrate alterations exhibited by LEC11 are then expressed on the N-linked biantennary
• the virus is purified by equilibrium gradient centrifugation, and
• glycopeptides are purified using proteinase digestion as described by Stanley et al.
• selectins Since the ligand is generally expressed on the cell surface of these cell types, one approach consists of isolating a plasma membrane fraction enriched in the ligand. Once plasma membranes have been isolated, the ligands may be isolated and subsequently identified using monoclonal antibodies,
• oligosaccharide structure such as monoclonal antibodies FH6, SNH3 and CSLEX-1.
• release of the oligosaccharide from the glycopeptide is generally the first step in the structural analysis of the oligosaccharide chain. This is accomplished by chemical cleavage of the proteincarbohydrate linkage, or by specifically releasing the
• O-linked carbohydrate units are released by alkaline ⁇ -elimination.
• the oligosaccharides are separated from the glycopeptides by gel filtration.
• the resulting oligosaccharides are then separated from each other using a combination of gel filtration, HPLC, thin layer chromatography, and ion exchange chromatography.
• the isolated oligosaccharides are then fully analyzed.
• Complete structural analysis of the purified oligosaccharide units requires the determination of the monosaccharide units, their ring form, configuration (D or L), anomeric linkage ( ⁇ or ⁇ ) , the positions of the linkages between the sugars and their sequence. In addition, the position of any substituent groups are established.
• Methylation analysis is used to determine the positions of the glycosidic linkages between the monosaccharides.
• the anomeric configuration of the sugar residues can be addressed using 500-MHz 1 H NMR spectroscopy.
• the conditions and methods used to perform a complete structural carbohydrate analysis are
• the state of the art techniques to fully characterize the sugars of an oligosaccharide include the use of several analytical techniques such as FAB-MS (fast atom bombardmentmass spectrometry), HPAE (high pH anion exchange
• One approach to characterizing the selectin ligand on glycolipids consists of disrupting the cells using organic solvents, isolating the glycolipids, and identifying those glycolipids reactive with monoclonal antibodies to SLX, such as FH6, SNH3, SNH4, CSLEX-1, or VIM-2, for example, and then determining the structure of the oligosaccharide chains.
• SLX monoclonal antibodies to SLX
• standard methods for glycolipid preparation can be used (see, e.g., Ledeen et al., J. Neurochem. 21:829 (1973), which is
• glycolipids are extracted from HL-60, HT-29, PMNs, human leukocytes, and other cell lines expressing the selectin ligand by methods generally known to those skilled in the arts (see, e.g.,
• glycosphingolipids that contain at least one sialic acid moiety are isolated and separated into neutral and acidic fractions using DEAE-Sephadex chromatography as described in detail by Ledeen and Yu, Methods Enzymol. 83:139 (1982). The resulting gangliosides are pooled, lyophilized, and dissolved in chloroform/methanol (2:1). The lower phase of the Folch partition contains glycolipids. These are isolated and
• the thin layer plate is then incubated with 1 25 I labeled FH6, or other monoclonal antibody which binds specifically to SLX. Following incubation with the labeled antibody, the plate is exposed to radiographic detection film and developed. Black spots on the X-ray film correspond to gangliosides that bind to the monoclonal antibody, and those gangliosides are recovered by scraping the corresponding areas of the silica plate and eluting the gangliosides with
• glycolipids are also dried and resuspended in chloroform and developed in a similar thin layer system and probed with the radiolabeled antibody. Structural analysis of oligosaccharides derived from glycolipids is performed essentially as described for glycoproteins.
• Oligosaccharides comprising the SLX unit can be prepared from glycoproteins by methods well known in the art (see, e.g., Gerard, supra, at pp. 537-539). Typically,
• N-glycosidase F (N-glycanase) is used to cleave N-linked oligosaccharides while O-linked groups are cleaved with endo-N-acetylgalactosaminidase.
• Synthetic compounds containing SLX or its mimetics attached to a variety of moieties can be prepared depending on the particular use desired.
• SLX can be converted to a ganglioside by linking a ceramide moiety to the C-1 of the reducing terminal GlcNAc unit.
• SLX structures can also be linked to a wide variety of other moieties such as variously substituted amino groups, heterocyclic compounds, ether
• the SLX unit may also be bound to various amino acids, amino acid mimetics, oligopeptides or proteins.
• alkyl as used herein means a branched or unbranched saturated or unsaturated hydrocarbon chain
• aryl refers to a radical derived from an aromatic hydrocarbon by the removal of one atom, e.g., phenyl from benzene.
• the aromatic hydrocarbon may have more than one unsaturated carbon ring, e.g., naphthyl.
• Heterocyclic compounds refers to ring compounds having three or more atoms in which at least one of the atoms is other than carbon (e.g., N, O, S, Se, P, or As). Examples of such compounds include furans, pyrimidines, purines, pyrazines and the like.
• SLX polyvalent forms of SLX
• monomeric units containing SLX can be joined to form molecules having one to about four or more SLX moieties.
• An example of such a polyvalent form is one in which the oligosaccharide units are linked by the following moieties:
• n and m are the same or different and are integers from 2 to 12; Y is O or S; and W is O, S, or NH; or
• Z is a 5- to 14-membered ring and the substituents on the ring are in a cis- or trans-relationship
• substituents are in a 1,2 to 1,(p/2)+1 arrangement, where p is the size of the ring. If the ring is a heterocyclic ring
• the oligosaccharide moieties are preferably linked to the nitrogen atoms on the ring.
• heterocyclic compounds that are suitable for this purpose include piperazine and homopiperazine.
• mimetics can be created by attaching the desired moiety to preformed carrier moieties with multiple sites of attachment. Examples include attachment of SLX to the amino groups of lysine and lysine-containing peptides, proteins, glycoproteins or the asparagine side-chain of such compounds.
• One method of preparing polyvalent forms of SLX is by addition of desired monosaccharide residues to
• polysaccharide which contains the linear core structure of SLX into a polyvalent SLX containing polysaccharide is achieved by enzymatic fucosylation.
• Native polysaccharide type la obtained from Group B Streptococcus is preferably used.
• the entire 200,000 dalton polysaccharide can be used for this purpose as well as fragemnts thereof.
• polysaccharides having a molecular weight between about 5,000 and about 300,000 can be used.
• a molecular weight between about 25,000 and about
• polysaccharide type la may be fucosylated for the
• polysaccharide to have activity typically, between about 5 and about 200 side chains are fucosylated, preferably between about 50 and about 150 are fucosylated.
• the synthesis of the SLX moiety can be accomplished using chemical, enzymatic, or combined chemical and enzymatic strategies. (see, e.g., EPO Publication No. 319,253, which is incorporated herein by reference.) In a preferred method
• a compound containing one or more N-acetylglucosamine units (GlcNAc-R) can be reacted sequentially with a galactosyltransferase (N-acetylglucosamine ⁇ 1,4
• Gal ⁇ 1, 3GalNAc ⁇ 2,3 sialytransferase (E.C. 2.4.99.4) and a fucosyltransferase (N-acetylglucosaminide ⁇ 1,3
• R may be a carrier moiety or activatable intermediate that will allow attachment to a suitable carrier moiety.
• Each enzymatic reaction uses the appropriate nucleotide sugar as a donor substrate to generate the following intermediates in the synthesis of SLX.
• the glycosyl transfer reactions may optimally be carried out with added alkaline phosphatase (e.g., from calf intestine, CIAP) to consume the nucleoside phosphate byproduct which may inhibit the reaction.
• galactosyltransferase and the sialyltransferase(s) are purified from natural sources (Beyer et al., supra. and
• Fucosyltransferases may also be identified from natural sources, as generally described in Crawley and Hindsgaul, Carbohyd. Res. 193:249-256 (1989), incorporated by reference herein.
• the enzyme can be cloned and
• RNA can be subtracted from the wild type CHO cells and LEC11 cells as described by Chirgwin et al., Biochemistry 18:5214-5299 (1979), and the poly A+ RNA isolated by chromatography on oligo(dT)- cellulose.
• cDNA from the LEC-11 cells can be prepared as described by Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Ed. (1989), Cold Spring Harbor Press, New York, which is incorporated herein by reference. The cDNA can be subtracted using the method of Davis (Handbook of Experimental Immunology, Vol. 2, pp.
• a cDNA library can then be constructed in the CDM8 expression vector using the subtracted cDNA (Seed,
• fucosyltransferase can be isolated using the expression cloning method described by Larsen et al., Proc. Natl. Acad.
• the full-length clone of the fucosyltransferase can then be used to produce a soluble recombinant enzyme as taught by Colley et al., supra.
• SLX Another source of SLX is ⁇ 1 -acid glycoprotein, which is a plasma glycoprotein, the carbhydrate moities of which can be fucosylated to produce SLX (see, Alpha.-Acid glycoprotein: Genetics. Biochemistry. Physiological Functions, and
• Gal ⁇ 1,4 (Fuc ⁇ 1, 3)GlcNAc (A). Reaction of this intermediate with an appropriate glycosyl acceptor (e.g., an alcohol moiety) results in compound (B).
• an appropriate glycosyl acceptor e.g., an alcohol moiety
• Modified fucosides may be included in the synthetic schemes to provide for SLX analogues which vary in this moiety.
• ⁇ -D-arabinosyl glycosides may be synthesized following known procedures, Nicolaou et al., J.Amer. Chem.Soc. 112:3693-3695 (1990) through the use of tri-O-benzyl arabinosyl halides.
• Other C-5 aryl or alkyl substituted arabinosyl moieties may be synthesized, Danishefsky et al.,
• R can also be aryl, substituted aryl (e.g., Me, OH, I; alone or in combination including 125 I), alkylaryl, arylalkyl or other moiety, as the skilled artisan would include for the desired use.
• the introduction of iodine into phenolic compounds such as tyrosine is known in the art. Radical groups containing phenols are useful for the introduction of 125 I radioisotope, yielding compounds which are useful in diagnosis.
• the SLX ligand as disclosed here may also be used to assay for the presence of compounds which are capable of inhibiting intercellular adhesion mediated by selectins.
• a number of methods can be used to assay the biological activity of test compounds for the ability to inhibit the selectinmediated response. Ideally, the assays of the present
• the agent or test compound to be screened will typically be a synthetic or naturally-produced biomolecule, such as a peptide, polypeptide, protein (e.g., monoclonal antibody), carbohydrate (e.g., oligosaccharide),
• a synthetic or naturally-produced biomolecule such as a peptide, polypeptide, protein (e.g., monoclonal antibody), carbohydrate (e.g., oligosaccharide),
• glycoconjugate nucleic acid, and the like.
• the compounds are synthetically produced using, for instance, the methods for synthesizing oligosaccharides described above (see, also,
• Test compounds may also be isolated from any natural source, such as animal, plant, fungal, or bacterial cells in accordance with standard procedures as described above. Potentially useful monoclonal antibodies can be prepared according to standard methods described in more detail, below.
• the assays of the present invention are particularly useful in identifying compounds which act as antagonists or agonists of a ligand molecule.
• Antagonists are compounds which reverse the physiological effect of a ligand or exclude binding of the ligand to the receptor.
• An antagonist usually competes directly or indirectly with the ligand for the receptor binding site and, thus, reduces the proportion of ligand molecules bound to the receptor.
• an antagonist will be the topographical equivalent of the natural ligand and will compete directly with the ligand for the binding site on the selectin.
• SLX mimetic is a molecule that conformationally and functionally serves as substitute for an SLX moiety in that it is recognized by a selectin receptor. Alternatively, if the ligand and the test compound can bind the receptor simultaneously, the
• the assays of the present invention can be used to identify synthetic or naturally occurring agonists, that is, compounds which bind the receptor and initiate a physiological response similar to that of the natural ligand.
• selectin receptor genes have been cloned, thus the genes can be inserted and expressed in a wide variety of cells, such as COS cells, CHO cells and the like.
• cells which do not normally express SLX are capable of being transformed with one or more glycosy1transferase genes which confer on the transformed cells the ability to synthesize the ligand. (see, e.g., Lowe et al., Cell 63:475-484 (1990), which is incorporated herein by reference.)
• the test compound or agent is incubated with labelled SLX-bearing cells and activated endothelial cells immobilized on a solid surface.
• Inhibition of cellular adhesion is then determined by detecting label bound to the surface after appropriate washes.
• promyelocytic HL-60 cells and activated human endothelial cells or activated platelets are used.
• isolated ligand molecules can also be used in the assays.
• isolated selectin-binding agent or "isolated SLX moiety” as used herein refer to a selectin binding or SLX-bearing compound that is in other than its native state, e.g., not associated with the cell membrane of a cell that normally expresses the ligand.
• an isolated SLX moiety may be a component of an isolated molecule, such as an oligosaccharide or a glycoconjugate.
• the isolated molecule may be synthesized or prepared from the membranes of SLX-bearing cells.
• the isolated selectin-binding agent or SLX moiety may be associated with a liposome or attached to a solid surface before use in the assay.
• Methods for preparing SLX-bearing liposomes and for immobilizing various biomolecules are extensively discussed below.
• competition assays which detect the ability of a test compound to competitively inhibit binding of a compound known to bind either the receptor or the ligand. Inhibition of binding between SLX and a selectin receptor is usually tested. Inhibition of other binding interactions are also suitable, for instance, inhibition of the binding between a monoclonal antibody (e.g., FH6) and SLX or between an SLX mimetic and a selectin inhibitor can be used. Numerous types of competitive assays are known (see, e.g., U.S. Patents No. 3,376,110,
• the assays of the present invention are also suitable for measuring binding of a test compound to one component alone rather than using a competition assay. For instance,
• immunoglobulins can be used to identify compounds that contain the SLX moiety.
• Standard procedures for monoclonal antibody assays, such as ELISA, may be used (see. Harlow and Lane,
• assay formats involve the detection of the presence or absence of various physiological changes in either ligand-bearing or selectin-bearing cells that result from the interaction.
• suitable assays include the
• any component of the assay including the ligand, the receptor, or the test compound, may be bound to a solid
• the solid surface may be a membrane (e.g., nitrocellulose), a microtiter dish (e.g., PVC or polystyrene) or a bead.
• the desired component may be covalently bound or noncovalently attached through unspecific bonding.
• organic and inorganic polymers both natural and synthetic may be employed as the material for the solid surface.
• Illustrative polymers include polyethylene, polypropylene, poly(4-methylbutene), polystyrene,
• polymethacrylate poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, etc.
• Other materials which may be employed include paper, glasses, ceramics, metals, metalloids, semiconductive materials, cermets or the like.
• substances that form gels such as proteins, e.g., gelatins, lipopolysaccharides, silicates, agarose and polyacrylamides or polymers which form several aqueous phases, such as dextrans, polyalkylene glycols
• alkylene of 2 to 3 carbon atoms or surfactants e.g.
• amphiphilic compounds such as phospholipids, long chain (12-24 carbon atoms) alkyl ammonium salts and the like. Where the solid surface is porous, various pore sizes may be employed depending upon the nature of the system.
• a plurality of different materials may be employed, particularly as laminates, to obtain various properties.
• protein coatings such as gelatin can be employed to avoid non-specific binding, simplify covalent conjugation, enhance signal detection or the like.
• the surface will usually be polyfunctional. or be capable of being polyfunctionalized.
• Functional groups which may be present on the surface and used for linking can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like.
• the manner of linking a wide variety of compounds to various surfaces is well known and is amply illustrated in the literature. See for example Immobilized Enzymes, Ichiro
• Noncovalent binding is typically nonspecific absorption of a compound to the surface.
• the surface is blocked with a second compound to prevent nonspecific binding of labelled assay components.
• the surface is designed such that it nonspecifically binds one component but does not significantly bind another. For example, a surface bearing a lectin such as Concanavalin A will bind a
• labelled assay components such as SLX ligands, SLX mimetics, immunoglobulins, receptors, or test compounds.
• the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. A wide variety of labels may be used.
• the component may be labelled by any one of several methods. The most common method of detection is the use of autoradiography with 3 H, 125 I, 35 S, 14 C, or 32 P labelled compounds or the like. The choice of radioactive isotope depends on research preferences due to ease of synthesis, varying
• nonradioactive labels include ligands which bind to labelled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labelled ligand.
• the choice of label depends on sensitivity required, ease of conjugation with the compound, stability requirements, and available instrumentation.
• Non-radioactive labels are often attached by indirect means.
• a ligand molecule e.g., biotin
• ligand covalently bound to the molecule.
• the ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
• a signal system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
• Ligands and anti-ligands may be varied widely. Where a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labelled, naturally occurring anti ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.
• the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
• Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
• Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
• Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
• luciferin 2,3-dihydrophthalazinediones
• luminol 2,3-dihydrophthalazinediones
• the present invention also provides monoclonal antibodies capable of inhibiting intercellular adhesion mediated by selectins as well as methods for identifying such antibodies.
• the monoclonal antibodies bind a selectin ligand or receptor and block cellular adhesion.
• immunoglobulin refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes.
• the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad
• immunoglobulin variable region genes may exist in a variety of forms besides antibodies, including for example, Fv, Fab, and F(ab) 2 , as well as in single chains
• Antibodies which bind the SLX antigen may be produced by a variety of means.
• the production of non-human monoclonal antibodies, e.g., murine, lagomorpha, equine, etc., is well known and may be accomplished by, for example, immunizing the animal with the SLX antigen or a preparation containing a glycoprotein or glycolipid comprising the antigen.
• Antibodyproducing cells obtained from the immunized animals are well known and may be accomplished by, for example, immunizing the animal with the SLX antigen or a preparation containing a glycoprotein or glycolipid comprising the antigen.
• human monoclonal antibodies to a human antigen in the case of an SLX unit isolated from human tissue
• a human antigen in the case of an SLX unit isolated from human tissue
• Such methods are generally known in the art and are described in, for example, U.S. 4,816,397, EP
• a number of currently available monoclonal antibodies can be used according to the present invention to inhibit intercellular adhesion mediated by selectins.
• selectins For instance, CSLEX-1 (see. Campbell et al., J. Biol. Chem. 259:11208-11214 (1984)), VIM-2, which recognizes a sequence slightly different from SLX (see. Macher et al., supra), FH6 (described in U.S. Patent No. 4,904,596) (all references are incorporated herein by reference) or SH 3 and SH 4 generated by Dr. S. Hakomori of the
• the compounds of the present invention can be used in preparing pharmaceutical formulations as discussed below. If the compound is an
• the SLX or SLX-mimetic moiety can be presented in a variety of forms, but should be able to effectively bind to a selectin receptor, such as ELAM-1, GMP-140, or MEL-14 antigen and thereby inhibit intercellular adhesion.
• a selectin receptor such as ELAM-1, GMP-140, or MEL-14 antigen
• compositions of the present invention can be used to block or inhibit cellular adhesion associated with a number of disorders. For instance, a number of inflammatory disorders are associated with selectins
• a specific defense system reaction is a specific immune system reaction to an antigen.
• Example of specific defense system reactions include antibody response to antigens, such as viruses, and delayed-type hypersensitivity.
• a non-specific defense system reaction is an inflammatory response mediated by leukocytes generally incapable of immunological memory. Such cells include macrophages, eosinophils and neutrophils. Examples of non-specific reactions include the immediate swelling after a bee sting, and the collection of PMN leukocytes at sites of bacterial infection (e.g., pulmonary infiltrates in bacterial pneumonias and pus formation in abscesses).
• treatable disorders include, e.g., rheumatoid arthritis, post-ischemic leukocyte-mediated tissue damage
• leukocyte-mediated lung injury e.g., adult respiratory
• atopic dermatitis psoriasis
• inflammatory bowel disease Various platelet-mediated pathologies such as atherosclerosis and clotting can also be treated.
• tumor metastasis can be inhibited or prevented by inhibiting the adhesion of circulating cancer cells. Examples include carcinoma of the colon and melanoma.
• compositions of the present invention are particularly amenable to treatment by compositions of the present invention.
• compositions which inhibit a GMP-140 selectin-ligand are particularly amenable to treatment by compositions of the present invention.
• interaction may be particularly useful for treating or
• the present invention may be used prophylactically prior to heart surgery to enhance postsurgical recovery.
• GMP-140 is stored in Weibel-Palade bodies of platelets and endothelial cells and is released upon activation by thrombin to mediate adhesion of neutrophils and monocytes
• inhibitors of the GMP-140 -ligand interaction may be especially useful in minimizing tissue damage which often accompanies thrombotic disorders.
• such inhibitors may be of therapeutic value in patients who have recently experienced stroke, myocardial infarctions, deep vein thrombosis, pulmonary embolism, etc.
• the compounds are especially useful in prethrombolytic therapy.
• compositions of the invention find particular use in treating the secondary effects of septic shock or disseminated intravascular coagulation (DIC).
• DIC disseminated intravascular coagulation
• the therapeutic compositions provided herein inhibit leukocyte emigration at these sites and mitigates tissue damage.
• the inhibitors of selectin-ligand interaction also are useful in treating traumatic shock and acute tissue injury associated therewith. Because the selectins play a role in recruitment of leukocytes to the sites of injury, particularly ELAM-1 in cases of acute injury and inflammation, inhibitors thereof may be administered locally or systemically to control tissue damage associated with such injuries. Moreover, because of the specificity of such inhibitors for sites of inflammation, e.g., where ELAM-1 receptors are expressed, these compositions will be more effective and less likely to cause complications when compared to traditional anti-inflammatory agents.
• the present invention also provides
• compositions which can be used in treating the aforementioned conditions.
• the pharmaceutical compositions are comprised of biomolecules or other compounds which comprise an SLX unit, antibodies which bind to SLX, or other compounds which inhibit the interaction between the SLX ligand and selectin receptors, together with pharmaceutically effective carriers.
• a biomolecule of the present invention may be a peptide, polypeptide, protein (e.g., an immunoglobulin), carbohydrate (e.g., oligosaccharide or polysaccharide),
• glycoconjugate e.g., glycolipid or glycoprotein
• nucleic acid e.g., nucleic acid, and the like.
• the pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see, Langer, Science 249:1527-1533 (1990), which is incorporated herein by reference.
• compositions of the present invention may comprise SLX bearing compounds in admixture with other
• the SLX ligand of the pharmaceutical composition can be used to target conventional anti-inflammatory drugs or other agents to specific sites of tissue injury.
• a selectin-binding oligosaccharide moiety such as an SLX ligand or SLX mimetic
• a drug to a selectin receptor on, e.g., a vascular endothelial cell
• a selectin-binding oligosaccharide moiety such as an SLX ligand or SLX mimetic
• chemotherapeutic agents can be substantially alleviated by the lower dosages, the localization of the agent at the injury sites and/or the encapsulation of the agent prior to delivery.
• the targeting component i.e., the SLX ligand or an SLX mimetic which binds to a desired selectin
• the coupling which may be performed by means, generally known in the art, should not substantially inhibit the ability of the ligand to bind the receptor nor should it substantially reduce the activity of the chemotherapeutic agent.
• a variety of chemotherapeutics can be coupled for targeting.
• anti-inflammatory agents which may be coupled include SLX- bearing compounds of the present invention, immunomodulators, platelet activating factor (PAF) antagonists, cyclooxygenase inhibitors, lipoxygenase inhibitors, and leukotriene
• Some preferred moieties include cyclosporin A, indomethacin, naproxen, FK-506, mycophenolic acid, etc.
• anti-oxidants e.g., superoxide dismutase
• anticancer agents can be targeted by coupling the SLX ligand or mimetic to the chemotherapeutic agent. Examples of agents which may be coupled include
• daunomycin daunomycin, doxorubicin, vinblastine, bleomycin, etc.
• the selectin receptor targeting may also be accomplished via amphipaths, or dual character molecules
• polar nonpolar
• Amphipaths include nonpolar lipids, polar lipids, mono- and diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids and salts. These molecules can exist as emulsions and foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions and lamellar layers.
• liposomes are generically referred to herein as liposomes.
• liposomes filled with a desired chemotherapeutic agent can be directed to a site of tissue injury by the selectin-SLX ligand interaction.
• the liposomes When the liposomes are brought into proximity of the affected cells, they deliver the selected therapeutic compositions.
• the liposomes of the present invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
• the selection of lipids is generally guided by consideration of, e.g., liposome size and stability of the liposomes in the bloodstream.
• the major lipid component in the liposomes is phosphatidylcholine.
• Phosphatidylcholines having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. In general, less saturated
• phosphatidylcholines are more easily sized, particularly when the liposomes must be sized below about 0.3 microns, for purposes of filter sterilization. Methods used in sizing and filter-sterilizing liposomes are discussed below.
• the acyl chain composition of phospholipid may also affect the stability of liposomes in the blood.
• One preferred phosphatidylcholine is partially hydrogenated egg phosphatidylcholine.
• targeting agents e.g., ligands, receptors and monoclonal antibodies
• ligands e.g., ligands, receptors and monoclonal antibodies
• Glycoproteins and glycolipids of a variety of molecular weights can be used as targeting agents. Typically, glycoproteins having a molecular weight less than about 300,000 daltons, preferably between about 40,000 and about 250,000 are used, more preferably between about 75,000 and about 150,000. Glycolipids of molecular weight of less than about 10,000 daltons, preferably between about 600 and about 4,000 are used. Standard methods for coupling targeting agents to liposomes can be used. These methods generally involve
• Antibody targeted liposomes can be constructed using, for instance, liposomes which
• Targeting mechanisms generally require that the targeting agents be positioned on the surface of the liposome in such a manner that the target agents are available for interaction with the selectin receptor.
• the liposome is typically fashioned in such a way that a connector portion is first incorporated into the membrane at the time of forming the membrane.
• the connector portion must have a lipophilic portion which is firmly embedded and anchored in the membrane. It must also have a hydrophilic portion which is chemically available on the aqueous surface of the liposome.
• the hydrophilic portion is selected so that it will be chemically suitable to form a stable chemical bond with the targeting agent which is added later.
• the connector molecule must have both a lipophilic anchor and a hydrophilic reactive group suitable for reacting with the target agent and holding the target agent in its correct position, extended out from the liposome' s surface.
• a lipophilic anchor and a hydrophilic reactive group suitable for reacting with the target agent and holding the target agent in its correct position, extended out from the liposome' s surface.
• Liposome charge is an important determinant in liposome clearance from the blood, with negatively charged liposomes being taken up more rapidly by the
• Liposomes with prolonged circulation half-lives are typically desirable for therapeutic and diagnostic uses. Liposomes which can be maintained from 8, 12, or up to 24 hours in the bloodstream provide sustained release of the selectin-ligand inhibitors of the invention, or may facilitate targeting of the inhibitors (which may be labelled to provide for in vivo diagnostic imaging) to a desired site before being removed by the reticuloendothelial system.
• the liposomes are prepared with about 5-15 mole percent negatively charged phospholipids, such as
• phosphatidylinositol Added negatively charged phospholipids, such as phosphatidylglycerol, also serves to prevent
• Membrane-rigidifying agents such as sphingomyelin or a saturated neutral
• phospholipid at a concentration of at least about 50 mole percent, and 5-15 mole percent of monosialylganglioside, may provide increased circulation of the liposome preparation in the bloodstream, as generally described in U.S. Pat. No. 4, 837,028, incorporated herein by reference.
• the liposome suspension may include lipid-protective agents which protect lipids and drug
• Lipophilic free-radical quenchers such as
• alphatocopherol and water-soluble iron-specific chelators such as ferrioxianine, are preferred.
• lipids are dissolved in a suitable organic solvent or solvent system and dried under vacuum or an inert gas to form a thin lipid film. If desired, the film may be redissolved in a suitable solvent, such as tertiary butanol, and then lyophilized to form a more
• multilamellar vesicles can be shifted toward smaller sizes by hydrating the lipids under more vigorous agitation conditions or by adding solubilizing detergents such as deoxycholate.
• the hydration medium contains the targeted drug at a concentration which is desired in the interior volume of the liposomes in the final liposome suspension.
• the drug solution contains between 10-100 mg/ml in a buffered saline.
• concentration of the targeting SLX molecule or mimetic which binds a selectin is generally between about 0.1 - 20 mg/ml.
• the liposomes may be sized to achieve a desired size range and relatively narrow distribution of liposome sizes.
• One preferred size range is about 0.2-0.4 microns, which allows the liposome suspension to be sterilized by filtration through a conventional filter, typically a 0.22 micron filter.
• the filter sterilization method can be carried out on a high through-put basis if the liposomes have been sized down to about 0.2-0.4 microns.
• Extrusion of liposome through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing liposome sizes to a relatively well-defined size distribution.
• the suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved.
• the liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size.
• the initial sized liposome suspension may contain up to 50% or more drug and targeting agent in free (non-encapsulated) form. Therefore, to maximize the advantages of liposomal targeted drug, it is important to remove free drug and targeting agent from the final injectable suspension.
• the liposomes in the suspension are pelleted by high-speed centrifugation leaving free compound and very small liposomes in the supernatant.
• Another method involves concentrating the suspension by ultrafiltration, then resuspending the
• gel filtration can be used to separate large liposome particles from solute molecules.
• the liposome suspension is brought to a desired concentration for use in intravenous administration. This may involve resuspending the liposomes in a suitable volume of injection medium, where the liposomes have been concentrated, for example by centrifugation or ultrafiltration, or concentrating the suspension, where the drug removal step has increased total suspension volume.
• the suspension is then sterilized by filtration as described above.
• the liposomeligand preparation may be administered parenterally or locally in a dose which varies according to, e.g., the manner of administration, the drug being delivered, the particular disease being treated, etc.
• compositions which comprise the SLX ligand, and/or SLX mimetics which bind to selectin
• the dose of the compound will vary according to, e.g., the particular compound, the manner of administration, the particular disease being treated and its severity, the overall health and condition of the patient, and the judgment of the prescribing physician.
• the dose is in the range of about 50 ⁇ g to 2,000 mg/day for a 70 kg patient.
• therapeutic administration should begin as soon as possible after the myocardial infarction or other injury.
• the pharmaceutical compositions are intended for parenteral, topical, oral or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment.
• the pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For
• unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and dragees.
• compositions for intravenous administration which comprise a solution of the compound dissolved or suspended in a
• aqueous carrier preferably an aqueous carrier.
• aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
• compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
• auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
• the concentration of SLX ligand or mimetic which may be combined with other SLX ligands or mimetics to form a
• the cocktail may also comprise a monoclonal antibody which binds to selectin receptor, e.g., a monoclonal antibody to ELAM-1 or GMP-140, combined with the SLX ligand, a ligand mimetic or a monoclonal antibody to the ligand, so as to effectively inhibit the ligand-receptor interaction.
• the cocktail components may be delivered via liposome preparations.
• a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 25 mg of the compound.
• Actual methods for preparing parenterally administrable compounds will be known or apparent to those skilled in the art and are described in more detail in for example. Remington's
• conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
• mannitol lactose
• starch magnesium stearate
• sodium saccharin talcum
• cellulose glucose, sucrose, magnesium carbonate, and the like.
• pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more SLX ligands or mimetics of the invention, preferably about 20% (see, Remington's.
• the compounds are cyclopentyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-oethyl
• oligosaccharide ligands or mimetics are 0.05% - 30% by weight, preferably 1% - 10%.
• the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
• esters or partial esters of fatty acids containing from 6 to 22 carbon atoms such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic
• polyhydric alcohol or its cyclic anhydride such as, for example, ethylene glycol, glycerol, erythritol, arabitol, mannitol, sorbitol, the hexitol anhydrides derived from
• esters such as mixed or natural glycerides may be employed.
• the surfactant may
• composition constitute 0.1%-20% by weight of the composition, preferably 0.25-5%.
• the balance of the composition is ordinarily
• Liquefied propellants are typically gases at ambient conditions, and are condensed under pressure.
• suitable liquefied propellants are the lower alkanes containing up to 5 carbons, such as butane and propane; and preferably fluorinated or fluorochlorinated alkanes. Mixtures of the above may also be employed.
• a container equipped with a suitable valve is filled with the appropriate propellant, containing the finely divided compounds and surfactant. The ingredients are thus maintained at an elevated pressure until released by action of the valve.
• compositions containing the compounds can be administered for prophylactic and/or therapeutic treatments.
• compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications.
• An amount adequate to accomplish this is defined as "therapeutically effective dose.” Amounts effective for this use will depend on the severity of the disease and the weight and general state of the patient, but generally range from about 0.5 mg to about 2,000 mg of SLX oligosaccharide or SLX mimetic per day for a 70 kg patient, with dosages of from about 5 mg to about 200 mg of the compounds per day being more commonly used.
• compositions containing the compounds of the invention are administered to a patient susceptible to or otherwise at risk of a particular disease.
• a patient susceptible to or otherwise at risk of a particular disease is defined to be a "prophylactically effective dose.”
• prophylactically effective dose the precise amounts again depend on the patient's state of health and weight, but generally range from about 0.5 mg to about 1,000 mg per 70 kilogram patient, more commonly from about 5 mg to about 200 mg per 70 kg of body weight.
• compositions can be carried out with dose levels and pattern being selected by the treating physician.
• the pharmaceutical formulations should provide a quantity of SLX oligosaccharide or SLX mimetic of this invention sufficient to effectively treat the patient.
• the compounds may also find use as diagnostics.
• labeled compounds can be used to locate areas of inflammation or tumor metastasis in a patient
• compounds can be labeled with 125 I, 14 C, or tritium.
• LEC11, HL-60, HT-29, certain adenocarcinomas (colo 205 cells in particular), and polymorphonuclear leukocytes (PMN, neutrophils) contain a very specific ⁇ 1, 3-fucosyltransferase I, which is able to transfer fucose from GDP-fucose to the sialylated substrates NeuAc ⁇ 2, 3Gal ⁇ 1, 4GlcNAc or NeuGc ⁇ 2, 3Gal ⁇ 1, 4GlcNAc.
• Golgi apparatus-derived vesicle fractions are prepared by a modification of the procedure described by Balch et al., Cell, 39:405 (1984) which is incorporated herein by reference.
• the LEC11, HL-60, HT-29, PMN, colo 205 or other cell lines containing the ⁇ l, 3-fucosyltransferase I are grown in suspension to a density of approximately 5 ⁇ 10 5 cells/ml. Cells are harvested from the suspension culture by
• the resulting cell pellet from a 12 liter suspension (6 ⁇ 10 9 cells) is resuspended in 3 volumes (packed cell volume) of ice-cold 0.25M sucrose (w/v) solution containing Tris-Cl (10mM), pH 7.0, heat inactivated fetal calf serum (7%), and Aprotinin (100 ⁇ g/ml, Sigma Chemical, Co. St. Louis, Mo.).
• the cells are disrupted (approximately 60 strokes) with a tight fitting Wheaton glass dounce homogenizer using the A pestle.
• the homogenate is centrifuged for 5 min. at 500 ⁇ g in a table-top clinical centrifuge. Lipid and insoluble material remaining at the top of the solution in the centrifuge tube is discarded.
• the cloudy supernatant is transferred to a clean tube, and the sucrose concentration of the supernatant fraction is then adjusted to 40% (w/v) sucrose in Tris-Cl (20 mM), pH 7.0, with the aid of a refractometer.
• sialytransferase is only known to be found within Golgi apparatus-derived vesicles and is used by those trained in the art as a marker to assess the authenticity of the band collected from the 29-35% interphase.
• Sialyltransferase assays are performed using asialofetuin as the acceptor as described by Briles et al., J. Biol. Chem., 252:1107 (1977).
• a good Golgi apparatus derived vesicle preparation from LEC cells typically has a sialyltransferasespecific activity of 3.0 nmole/mg protein/hr.
• the resulting Golgi apparatus preparation is then used as a source of the ⁇ 1, 3-fucosyltransferase I used in the enzymatic synthesis described above.
• LEC11 cells which express SLX
• ELAM-1 activated endothelial cells expressing ELAM-1
• LEC12 glycosylation mutant
• LEC11, LEC12 and CHO-K1 were provided by Dr. P. Stanley. They were grown in suspension culture in complete alpha MEM
• HL-60, LEC11, LEC12 and CHO-K1 cells were harvested and washed in CRPMI. A viable cell count was made using trypan blue. 3 ⁇ 10 6 cells of each type were pelleted in a 10 ml test tube and 300 ⁇ l of 51 Cr (450 ⁇ Ci) (New England Nuclear) was added to each pellet. The tubes were allowed to incubate 1 hour at 37oC with gentle agitation. 2. Labeled cells were washed 3X in medium and resuspended to 2 ⁇ 10 5 / 400 ⁇ l (6ml). The tubes were then placed in a 4oC ice bath.
• the medium was removed from the assay wells with a pasteur pipette a few wells at a time.
• the plate was incubated in the ice water bath for 30 minutes.
• the P1000 tip was ejected into the tube. 10.
• the tubes, including those containing the input CPM samples were counted in a gamma counter.
• HUVEC Passage 3 HUVEC from cultures initiated for the present experiments were used as described above. Two sets of triplicate wells were left unstimulated as controls. Four triplicates were stimulated with IL-1 ⁇ (Genzyme) at 10 ⁇ g/ml and 4 at 20 ⁇ g/ml. Cells were stimulated for exactly 4 hours. HL-60 cells obtained from the American Type Culture Collection were used as the source of ligand bearing cells.
• CRPMI-1640 Gibco
• penicillin 100 units/ml
• streptomycin 100 ⁇ g/ml
• L-Glutamine 2mM
• Fetal Bovine Serum Hazleton
• Monoclonal antibody preparations included SNH3 (IgM) at about 20 ⁇ g/ml and SH1 (IgG3) at about 10 ⁇ g/ml.
• SNH3 SNH3
• SH1 IgG3
• the specificity of SNH3 is for SLX, while SH1 recognizes the unsialylated structure.
• HL-60 cells were harvested and washed in CRPMI.
• a viable cell count was made using trypan blue. 3 ⁇ 10 6 cells were placed in each of 2, 10 ml test tubes and 300 ⁇ l of 51 Cr (450 ⁇ Ci) (New England Nuclear) was added to each tube. The tubes were allowed to incubate 1 hour at 37oC with gentle agitation.
• 51 Cr 450 ⁇ Ci
• the antibodies were supplied as hybridoma culture supernatants and contained 0.01% NaN3 and 0.05%
• Antibodies were collected from dialysis and 3.5 ml of each was placed in 10 ml tubes. The remainder was retained for use in an ELISA assay for HL-60 binding. 7 ml of RPMI 1640 5% FCS was placed in a 4th tube for use as a control.
• the stimulated HUVEC assay plate was removed from the incubator and the medium was removed from the wells with a pasteur pipette, a few wells at a time. 7. 0.5 ml of cell suspension was added to each of triplicate wells. Control cells were plated on unstimulated and stimulated HUVEC at both IL-1 ⁇ concentrations. Test cells were added to stimulated wells only.
• Unbound cells were removed from the wells of the assay plate by systematic resuspension using a pasteur pipette followed by addition and removal of 0.7 ml of medium.
• the tubes, including those containing the input counts per minute (CPM) samples were counted in a gamma
• IgM a lower affinity mAb
• SNH-3 (20 ⁇ g/ml) (20 ⁇ g/ml
• SH-1 (10 ⁇ g/ml)
• FH-2 IgM
• SNH-4 IgG3
• CSLEX-1 IgM
• DMEM Dulbecco's Modified Eagles Medium
• the specificities of the antibodies were as follows: FH6, SNH-4, SNH3 and CSLEX-1 were specific for SLX; FH2 and SH1 were specific for the unsialylated Le x .
• HL-60 cells were harvested and washed in CRPMI. A viable cell count was made using trypan blue. 3 ⁇ 10 6 cells were placed in each of 2, 10 ml test tubes and 300 ⁇ l of 51 Cr (450 ⁇ Ci) (New England Nuclear) was added to each tube. The tubes were allowed to incubate 1 hour at 37oC with gentle agitation. 2. Labeled HL-60 cells were washed 3X in DMEM containing 5% FCS (hereafter referred to as cDMEM) and pooled into one tube. They were then centrifuged and resuspended to 4 ⁇ 10 6 cells per ml in the same medium.
• cDMEM 5% FCS
• the assay plate was incubated at 37oC for 30 min.
• the cell suspensions were plated on the chilled plate as for the 37oC plate above. This plate was incubated for 30 min. at 40oC. The remaining steps of the assay were performed as described in steps 11-15 of Section A above, except that in step 12 the plates were allowed to stand for 15 min. rather than 30 min.
• Fig. 2A The results, shown in Fig. 2A, indicate that the monoclonal antibodies SNH-3, FH6, SNH-4 and CSLEX-1, all specific for SLX, significantly blocked the binding of HL-60 cells to IL-1 ⁇ stimulated HUVEC via the ELAM-1 receptor when incubated at 37°C.
• the monoclonal antibodies specific for Le x (FH2 and SH1) were not effective inhibitors.
• the ligand for ELAM-1 contains the sialylated Le x antigen or a similar structure found in cell surface glycoproteins or glycolipids.
• LEC11 and LEC12 cells were harvested and washed in CRPMI. A viable cell count was made using trypan blue. 3 ⁇ 10 6 cells of each cell line were placed in each of 2, 10 ml test tubes and 300 ⁇ l of 51 Cr (450 ⁇ Ci) (New England Nuclear) was added to each tube. The tubes were allowed to incubate 1 hr. at 37oC with gentle agitation.
• the radiolabeled cells were washed X3 in cDMEM and pooled into one tube. They were then centrifuged and resuspended to 4 ⁇ 10 6 cells per ml in the same medium.
• the stimulated HUVEC assay plate was removed from the incubator and the wells were washed one time with cDMEM and the medium was removed from the wells with a pasteur pipette, a few wells at a time. 6. The cell suspensions and the assay plate were placed in an ice bath to chill for 20 min.
• the assay plate was incubated for 30 min. at
• step 12 The remaining steps of the assay were performed as described in steps 11-15 of Section A, above, except that in step 12 the plates were allowed to stand for 15 min.
• Liposomes of Glycosphingolipids Block Binding of SLX Cells to Activated Endothelial Cells This Example describes the preparation of liposomes which contain various biosynthetically produced
• glycosphingolipids on which the terminal carbohydrate units are either SLX, Le x , or similar but not identical compounds.
• the ability of the liposomes which contain SLX or SLX mimetics to block the binding of SLX-expressing HL-60 cells and LEC11 cells to endothelial cells which have been stimulated to express ELAM-1 by treatment with IL-1 ⁇ is shown.
• Materials: The glycosphingolipids used in this experiment are shown in Table I; they were obtained from the Biomembrane Institute, Seattle, WA, and were either purified or biosynthetically produced and characterized by NMR and mass spectrometry, as generally described in Hakomori, S. I., et al., J. Biol.
• S-diLe x (SLX) was synthesized enzymatically by adding fucosyl residues using a colo 205 cell line as enzyme source and SH as substrate. Nonsialylated diLe x was similarly
• nLc6 synthesized using nLc6 as substrate and the cell line NCI H-69. See Holmes et al., J. Biol. Chem. 260:7619 (1985), incorporated by reference herein. SPG and SH were purified form bovine red blood cells, and nLc6 was produced by chemical removal of the terminal sialosyl residue from SH.
• Liposomes containing the glycosphingolipids were formed as follows: 100 ⁇ g of glycolipid was added to 300 ⁇ g phosphatidylcholine (Sigma, egg yolk) and 500 ⁇ g cholesterol (Sigma) in chloroform-methanol (2:1) and the whole solution evaporated to dryness by N 2 in 15 ml screwcap tubes.
• HL-60 cells and LEC11 cells were harvested and washed in CRPMI. A viable cell count was made using trypan blue. 6 ⁇ 10 6 cells of each cell type were radiolabeled as follows: 3 ⁇ 10 6 cells of each type were placed in each of 2, 10 ml test tubes and 300 ⁇ l of 51 Cr (450 ⁇ Ci) (New England Nuclear) was added to each tube. The tubes were allowed to incubate 1 hr. at 37oC with gentle agitation.
• 51 Cr 450 ⁇ Ci
• Radiolabeled HL-60 and LEC11 cells were washed 3X in CRPMI and pooled into one tube. They were then
• the medium was removed from the wells of the assay plate with a pasteur pipette, a few wells at a time, and 0.3 ml of liposome suspension was added to each of 6 IL-1,5 stimulated assay wells.
• Control wells received the liposome buffer containing ethanol, RPMI 1640 and BSA at the same concentrations as in the liposome containing wells.
• Control buffer was plated on unstimulated and stimulated HUVEC.
• Liposome containing samples were added to stimulated wells only.
• liposomes containing selected glycolipids having terminal sequences which contained SLX dramatically inhibited adhesion of HL-60 cells to activated endothelial cells at 4'C.
• Liposomes containing glycolipids with Le x (di-Le x ) or other related carbohydrate structures (Table 1) exhibited minimal inhibition that was not dependent on the structure of the carbohydrate group. Similar results were obtained with LEC 11 cell adhesion.
• compositions can serve as therapeutic compounds for, e.g., the reduction of leukocyte infiltration into inflammatory sites.
• Jurkat cells bind to IL-1 activated endothelial cells predominantly through the V-CAM (endothelial cell) - VLA-4 (Jurkat cell) adhesion pair (Wayner et al., J. Cell Biol.
• HL-60 cells are described above and were used as the source of ligand bearing cells.
• Jurkat cells were used as the non-ligand bearing control.
• Monoclonal antibodies SH-1, FH-2, SNH-4, and CSLEX-1 are also described above.
• Blood was drawn from a normal human donor into a syringe containing ACD anticoagulant (dextrose, 2.0 g; sodium citrate 2.49 g; and citric acid 1.25 g; to 100 ml with dH 2 O) at a ratio of 6 parts blood to 1 part anticoagulant.
• ACD anticoagulant dextrose, 2.0 g; sodium citrate 2.49 g; and citric acid 1.25 g; to 100 ml with dH 2 O
• centrifugation as follows: Blood was centrifuged at 800 rpm (approx. 90 ⁇ g) for 15 min. at room temp. The supernatant was collected and centrifuged at 1200 rpm (approximately 400 ⁇ g) for 6 min. The supernatant was removed and centrifuged at 2000 rpm (1200 ⁇ g) for 10 min. to pellet the platelets.
• the platelet button was washed 2 times with Tyrode-HEPES buffer, pH 6.5 (NaCl 8.0 g; KCl 0.2 g; NaH 2 PO 4 H 2 O 0.057 g; MgCl 2 6H 2 O 0.184 g; NaHCO 3 0.1 g; Dextrose, 1.0 g; and HEPES, 2.383 g; bring to 1 L with DI water, adjust to pH 6.5 with 1N NaOH) followed by one wash in PBS. Platelets were suspended to a concentration of 10 8 /ml in PBS.
• Platelets were activated by the addition of 0.25 units of thrombin/ml (Sigma T-6759) of platelet suspension. Platelets were allowed to stand at room temperature for 20 min.
• HL-60 and Jurkat cells were harvested and washed in CRPMI. A viable cell count was made using trypan blue, 3 ⁇
• Radiolabeled cells were washed 3X in CRPMI and pooled into one tube. They were then centrifuged and
• the assay plate was centrifuged at 90 xg for 2 min. and then incubated for 5 min. at room temp. Unbound cells were removed from the wells of the assay plate by inverting the plate into a radioactive waste receptacle and blotting the plate on towels. The wells were washed X3 by carefully adding 300 ⁇ l PBS to each well and inverting and blotting the plate. All of the medium was removed from the wells and 0.3 ml of a solution of 0.125 M Tris, 2% SDS and 10% glycerin was added. The plates were allowed to stand for 15 min. and then 0.6 ml of dH 2 O was added to each well.
• Glycosphingolipids were prepared as described in Example IV.
• the platelets were prepared as described in
• HL60 cells were prepared as described above.
• PMNs were prepared from 50 ml of whole blood drawn from volunteer donors into heparinized vacutainer tubes, which were inverted to mix the blood. All steps were performed at 22-24 degrees C. Each 25 ml of blood was layered over 15 ml of Mono-Poly Resolving Medium (Flow Labs). The tubes were
• the platelets were incubated with the liposome preparations for 20 min. at room temp.
• Neutrophils or HL-60 cells at 2 ⁇ 10 6 cells/ml were each added to one set of liposome treated platelets.
• the tubes were mixed and allowed to stand at room temperature for 20 min. Then they were applied to a hemacytometer and the cells were scored as positive (2 or more platelets attached/cell) or negative (less than 2 platelets attached/cell).
• liposomes containing selected glycolipids having terminal sequences which contained SLX dramatically inhibited adhesion of HL-60 cells to activated platelets.
• Liposomes containing glycolipids with Le x (di-Le x ) or other related carbohydrate structures (Table 1) exhibited minimal inhibition that was not dependent on the structure of the carbohydrate group. Similar results were obtained with PMN cell adhesion (Fig. 8).
• Hexasaccharide SLX blocks binding of Neutrophils to platelets
• a minimal tetrasaccharide SLX to inhibit GMP-140 adhesion was compared to that of a hexasaccharide SLX.
• platelets and neutrophils were isolated by the methods described above. Platelets were activated with thrombin and then incubated with dilutions of various oligosaccharides. Neutrophils were added and the effect of the saccharides on the adhesion of neutrophils to activated platelets was determined.
• the oligosaccharides used were as follows: SLX(hexa), NeuAc ⁇ 2, 3Gal,91,4 (Fuc ⁇ 1, 3)
• glycolipid-liposome preparations prepared as described, above, at concentrations from 2 ⁇ g/ml to 0.25 ⁇ g/ml, was added and the tubes were allowed to stand at room
• Blocking adhesion using variant SLX structures This example describes experiments testing various glycolipid structures on liposomes.
• SY2 a sialylated polysaccharide in which the fucose instead of being attached to the ultimate GlcNAc as in SLX, is attached to the penultimate GLcNAc was tested. Platelets and neutrophils were isolated by the methods described above. Platelets were activated with thrombin and then incubated with dilutions of various glycolipids embedded in liposomes prepared as described above. Neutrophils were added and the effect of the
• terminal sialic acid is in the form N-Acetyl neuraminate
• NeuronAc N-Glycol neuraminate
• NeuroGc N-Glycol neuraminate
• All materials were prepared as described above. Platelets and neutrophils were isolated by the methods described. Platelets were activated with thrombin and then incubated with dilutions of various glycolipids contained in liposomes. Neutrophils were added and the effect of the glycolipids on the adhesion of neutrophils to activated platelets was determined.
• sialic acid was NeuAc or NeuGc. This result indicates that either the N-acetyl or N-glycollyl derivative of sialic acid would also allow
• This example also shows that the linkage of the sialic acid affects binding to ELAM-1.
• Two synthetic compounds were prepared. One comprised sialic acid in an ⁇ 2.3 linkage, as in naturally occurring SLX. The second comprised sialic acid in an ⁇ 2,6 linkage, to examine the importance of the nature of the linkage to receptor
• Liposomes were prepared by adding 12 ⁇ l of absolute ETOH to each tube, warming briefly in a 50oC water bath and sonicating for 2 min. 238 ⁇ l of warm phosphate buffered saline (PBS) was added slowly to each tube while sonicating and sonication was continued for a further 10 min. The final concentration of stock liposomes was 400 ⁇ g glycolipids/ml in 5% ETOH/PBS.
• PBS phosphate buffered saline
• HUVECs, PMNs, and liposomes were prepared as described
• Control buffer was plated on
• concentration of cells was 5 ⁇ 10 5 well in 100 ⁇ l.
• the assay plate was returned to the incubator (5% CO 2 , 37 oC) for 8 min. 8. Unbound cells were removed from the wells of the assay
• the plate was incubated on a rotary shaker for 10 min and then 0.05 ml of OPDA solution [8 mg o-phenylene-diamine, Sigma cat# P-1526, 8 ⁇ l of 30% H 2 O 2 and 10 ml of citrate buffer (as above)] was added to each well. The reaction was allowed to develop for 15 min and then 25 ⁇ l of 4N H 2 SO 4 was added to each well to stop the reaction. 11.
• a reagent bulk was prepared by mixing 100 ⁇ l volumes of the solubilization buffer and the OPDA solution with 50 ⁇ l of 4N H 2 SO 4 .
• Platelets were isolated and activated with thrombin by the methods described above. Cultured HL60 cells were treated with endo- ⁇ -galactosidase as described below and the effect of enzyme treatment on the GMP-140 mediated adhesion of HL60 cells to activate platelets was determined.
• Enzyme treatment of the HL60 cells was carried out as follows: 12.4 ⁇ 10 6 cells were washed twice with Hanks Balanced Salt Solution containing 20mM HEPES and 0.2% glucose, followed by a single wash step in normal saline.
• the endo- ⁇ -galactosidase (0.1 Unit, ICN Chemicals, Inc., Irvine, CA) was dissolved in 200 ⁇ l normal saline and 200 ⁇ l sodium acetate buffer, pH 6.01. 200 ⁇ l (containing 0.05U of enzyme) was added to 3 ⁇ 10 6 HL60 cells, and 200 ⁇ l of the acetate buffer was added to a similar number of cells to be used as the buffer control. Both tubes were incubated at 37oC for 60 min. with gentle shaking. The tubes were then cooled in ice and the cells were washed three times in HBSS containing HEPES and glucose and were then counted and suspended to 2 ⁇ 10 6 /ml.
• a hexasaccharide SLX and Le x compared to that of the non-fucosylated polysaccharide, a hexasaccharide SLX and Le x .
• platelets and neutrophils were isolated by the methods described above. Platelets were activated with thrombin and then incubated with dilutions of various oligosaccharides. Neutrophils were added and the effect of the saccharides on the adhesion of neutrophils to activated platelets was determined.
• the oligosaccharides used were as follows: Native polysaccharide and its fucosylated derivative (the preparation of both is described, below); SLX hexasaccharide, LNF III (Le x ) and LNF I (the structures are described above).
• the native type Ia polysaccharide 1 mg. was dissolved in a mixture of 6 ⁇ L of 1 M manganese chloride, guanosine 5'-diphosphate ⁇ -L-fucose with a radiolabelled tracer (specific activity 1.82 ⁇ 10 6 cpm/ ⁇ mol), 0.9 ⁇ moles in water 90 ⁇ L and water 137 ⁇ L.
• a radiolabelled tracer specific activity 1.82 ⁇ 10 6 cpm/ ⁇ mol
• Platelets were isolated as described above and were activated (2 ⁇ 10 8 /ml) by incubation for 20 min at room
• Neutrophils were isolated by layering heparinized blood over Mono-Poly Resolving Medium (Ficoll-Hypaque, Flow Laboratories), followed by centrifugation for 25 min at 2000rpm and then, a further 25 min at 2500rpm as described above.
• concentrations from 500 ⁇ g/ml to 2.0 ⁇ g/ml was added and the tubes were allowed to stand at room temperature for 20 min.
• neutrophils to thrombin activated platelets- 50% inhibition was achieved with less than l ⁇ g/ml. This compared to 20 ⁇ g/ml which was required of the native polysaccharide and 8 ⁇ g/ml of the SLX hexasaccharide for a similar degree of inhibition.

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