RU2123338C1 - Pharmaceutical composition based on intercellular adhesion mediator (variants), method of inhibition of intercellular adhesion, method of treatment of patients with inflammatory disease, method of analysis of the test-compound with respect to its capability to inhibit intercellular adhesion, method of compound synthesis - Google Patents

Abstract

FIELD: organic chemistry, medicine, pharmacy. SUBSTANCE: invention relates to compositions that have at least one component binding with selectin. Selectin-binding components have a general formula: R¹-Gal-1,4-(Fuc-α-1,3-GlcNac-R²)_a-X where R¹ is oligosaccharide or R³-R⁴-C(CO₂H) where R₃ and R₄ can be both similar and distinct - H, -C₁-C₈-alkyl, hydroxyl--C₁-C₈-alkyl, aryl-C₁-C₈-alkyl or alkoxy-C₁-C₈-alkyl where R² is β-1,3-Gal, α-1,2-Man or α-1,6-GalNAc; a = 0 or 1. Compositions can be used for attenuation or control over inflammation and for treatment of patients with inflammatory and other pathological disorders where intercellular adhesion takes part. EFFECT: broadened pattern of agents. 79 cl, 14 ex, 11 dwg, 6 tbl

Description

 The present invention relates to compositions and methods for reducing or suppressing inflammation, and for treating inflammatory processes and other pathological disorders in which intercellular adhesion is involved.

 Vascular endothelial cells and platelets (platelets) play a key role in several biological responses through the selective binding of certain cells, such as phagocytic leukocytes, in the blood stream. For example, endothelial cells preferably bind monocytes and granulocytes before they migrate through the wall of a blood vessel and into the surrounding tissue in an inflammatory reaction. It is known that some compounds that suppress inflammation act directly on the vascular endothelium in order to promote the adhesion of leukocytes to the walls of blood vessels. Then the cells pass through the walls to the area of damage or infection. It is assumed that cell adhesion to vascular endothelium also exists in tumor metastases. Circulating cancer cells obviously take advantage of the normal inflammatory mechanisms of the body and bind to areas of the walls of the blood vessels where the endothelium is activated.

 Blood plates also take part in the same reactions. It is known that blood plates become activated during the initiation of hemostasis and undergo major morphological, biochemical and functional changes (for example, rapid exocytosis of granules or degranulation), in which the membrane of the alpha granule of the plate merges with the outer plasma membrane. As a result, new cell surface proteins begin to be expressed, which give activated plates new functions, such as the ability to bind to both other activated plates and other cells. Activated platelets (platelets) are included in growing blood clots or quickly release circulating blood. Activated platelets are known to bind to phagocytic leukocytes, including monocytes and neutrophils. Examples of pathological and other biological processes in which the latter process is apparently involved as an intermediate include atherosclerosis, blood clot formation and inflammation.

 Recent work has revealed that specialized cell surface receptors on endothelial cells and platelets, referred to as leukocyte adhesion endothelial molecule-1 (ELAM-1) and granule membrane protein-140 (GMP-140), respectively, are included in the recognition of various circulating cells endothelium and platelets. These receptors are surface glycoproteins with a lectin-like region, a region with homology relative to the epidermal growth factor, and a region with homology with respect to additional regulatory proteins / cm. Bevilacgua et al., Science v. 243, p. 1160/1989 /, which is incorporated herein by reference /. For example, it was shown that ELAM-1 is involved in endothelial adhesion of leukocytes, which is the first stage in many inflammatory reactions. In particular, ELAM-1 binds to human neutrophils, monocytes, eosinophils, some T-lymphocytes / N. Graber et al., J. Jmmunol, T. 145, p. 819/1990 //, NK cells and the HL-60 line of promyelocytic cells.

 The term "selectin" has been proposed for a general class of receptors, which includes ELAM-1 and GMP-140, due to their region resembling lectin and the selective nature of their adhesive functions. These cell surface receptors are expressed on a wide variety of cells. GMP-140 (also known as PADGEM) is found on the surface of platelets and endothelial cells, where it is involved in the interactions of platelet-leukocyte and endothelial-leukocyte. Another member of the selectin class is the MEL-14 antigen and its human counterpart LAM-1, which are cell surface lymphocyte receptors and act as home lymphatic receptors. The exact nature of the ligand recognized by selectin receptors remains unknown.

 Various other methods have been previously developed to block the action of selectins and, thus, inhibit cell adhesion. For example, the use of monoclonal antibodies directed at ELAM-1 has been proposed as a procedure for inhibiting the adhesion of endothelial leukocytes to treat pathological reactions such as inflammation. Endothelial interleukin-8 has also been found to be an inhibitor of leukocyte-endothelium interactions.

 Once the ligand-receptor interaction is clarified, it will become possible to develop highly specific, effective cell adhesion inhibitors involving selectin that would be very useful in therapeutic treatment. A ligand (s) can also be used to synthesize other pharmaceutical compounds, such as anti-inflammatory agents or antioxidants, for use on lesion sites. To date, a lack of understanding of the interaction of ligands and receptor molecules on the corresponding cells has complicated these efforts. The present invention satisfies these and some other needs.

New compositions that selectively bind to a selectin receptor and which contain at least one selectin binding component are the subject of the present invention. Binding selectin constituents have the general formula:
R ₁ -Gal β 1,4 (Fue α 1,3) GlcNAc - (R ₂ ) _a ,
wherein
R ₁ is an oligosaccharide or R ₃ -R ₄ -C / CO ₂ H / -, in which R ₃ and R ₄ , which may be the same or different, are -H, C ₁ -C ₈ alkyl, -hydroxyl C ₁ -C ₈ alkyl, -aryl C ₁ -C ₈ alkyl or -alkoxy C ₁ -C ₈ alkyl;
in which R ₂ is β 1, 3Gal, α 1,2 Man, or α 1,6 GalNAc and a is 0 or 1.

In some preferred embodiments, R ₁ is sialic acid, generally NeuAc or NeuGc.

If R ₁ is an oligosaccharide, it is preferably Neu Ac α 2, 3 Gal β 1, 4GlcNAc β 1 or Neu Gc α 2, 3 Gal β 1, 4GlcNAc β 1,3. The selectin binding constituents are generally oligosaccharides containing a minimal tetrasaccharide (referred to as SLX) recognized by the selectin receptor.

 This compound is generally prepared from a polysaccharide having a repeating unit containing the core structure of unfucosylated SLX. With fucosylation, a polyvalent, SLX-bearing, polysaccharide is obtained. Preferred polysaccharides for this purpose are polysaccharides of type 1a, type II and type III from Group B streptococcus. Polyvalent selectin binding compounds are also prepared by combining selectin binding components with various linker components.

 The proposed compositions inhibit intercellular adhesion, in which the cell surface selectin receptor is involved, and while they are capable of, for example, inhibiting inflammation and other pathological reactions associated with cell adhesion. In appropriate embodiments, the composition that binds selectin may be a polysaccharide, glycoprotein, glycolipid or oligosaccharide.

 In accordance with the present invention, in particular, the aforementioned compounds in pharmaceutical compositions are provided. These pharmaceutical compositions may contain, for example, liposomes that contain a component capable of selectively binding to a selectin receptor and a pharmaceutically acceptable carrier. A liposome containing such a component can also serve as a “carrier to target” for a known chemotherapeutic agent that is enclosed within the liposome and is delivered to targeted cells that express a selectin receptor. In general, the chemotherapeutic agent is an anti-inflammatory agent or antioxidant. The use of the constituents described herein for delivering to the target chemical agents enclosed within liposomes is a known and effective technique for reducing therapeutic drug concentrations and minimizing side effects.

The pharmaceutical compositions of this invention may also contain immunoglobulins capable of selectively binding to an oligosaccharide ligand recognized by the selectin receptor. Suitable immunoglobulins for this purpose are CSLEX-1, FH ₆ , SNH ₃ , SNH ₄ and UTM-2.

 In other aspects, the present invention provides methods of inhibiting intercellular adhesion in a patient with a disease such as inflammation, by applying to the patient a therapeutically effective dose of a compound containing a component capable of binding to selectin receptor. A selectin receptor, such as ELAM-1 or GMP-140, can be expressed on vascular endothelial cells or platelets. The inflammatory process can be, for example, septic shock, a wound associated with sepsis, acute respiratory disease.

FIG. 1 illustrates the ability of cells that express SLX (LEC 11) to bind to IL-1β-activated endothelial cells compared to those cells that express non-sialylated Le ^x (CHO-K1 and LEC 12).

FIG. Figure 2 illustrates the ability of SLX-specific monoclonal antibodies to block HL-60 cell selectin binding at 37 ^° C (Fig. 2A) and 4 ^° C (Fig. 2B) compared to monoclonal antibodies that do not bind SLX determinants .

 FIG. 3 illustrates the incubation effects of LEC 11- (FIG. 3A) and LEC 12- of FIG. 3B) cells with SLX and non-SLX specific monoclonal antibodies for binding to activated endothelial cells.

 FIG. 4 illustrates the results obtained by treating HL-60, LEC11 and LEC12 cells with sialidase before binding to activated endothelial cells.

FIG. 5 compares the ability of liposomes that 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 platelet adhesion involving GMP-140 using monoclonal antibodies specific for SLX and Le ^x determinants.

FIG. 7 compares the ability of liposomes that 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 that contain glycolipids with SLX, Le ^x or similar carbohydrate structures to inhibit the binding of PMN to activated platelets.

 FIG. 9 shows inhibition of adhesion involving GMP-140 with glycolipids with terminal sialic acid, either NeuAc or NeuGc.

 FIG. 10 shows prophylactically applicable anti-ELAM-1 monoclonal antibodies to prevent lipopolysaccharide-induced death of rats.

 FIG. 11 shows therapeutically used anti-ELAM-1 monoclonal antibodies to prevent lipopolysaccharide-induced death of rats.

 Compositions and methods for inhibiting inflammatory and other painful reactions in which cell adhesion is involved are provided. The present invention also provides compounds (for example, glycoconjugates and monoclonal antibodies) that are capable of blocking or inhibiting cell adhesion involving cellular surface selectin receptors. Also provided are methods for the preparation and sieving of such compounds. A diagnostic and therapeutic use of such compounds is also proposed.

 The basis of the present invention is the discovery of a carbohydrate moiety recognized by surface selectin receptors. As mentioned above, selectins, also known as the "LEC-CAM" family of molecules in cell adhesion, are unique glycoproteins expressed on the surface of a wide variety of cells. For example, ELAM-1 is inducibly expressed on vascular endothelial cells / Bevilacgua et al., See above and Hession et al., Proc. Natl. Acad Sci., Vol. 87, pp. 1673-1677 / 1990 /; both of these works are incorporated herein by reference. This receptor has been found to be induced by inflammatory cytokines such as interleukin 1β1 (IL-1 β) and tumor necrosis factor α (TNFα, as well as bacterial endotoxin / lipopolysaccharide / / see Bevilacgua et al., Proc. Natl. Acad Sci., T. 84, pages 9238-9242 / 1987 /, which is incorporated herein by reference /. These compounds act directly on endothelial cells in vitro to significantly increase the adhesion of polymorphonuclear leukocytes (neutrophils) and monocytes (Bevilacgua and others ., Proc. Natl. Acad. Sci., See above).

 As already mentioned above, GMP-140 is a membrane glycoprotein of platelets and endothelial secretory granules / Geng and others, Nature T. 343, pages 757-760 / 1990 /, which is incorporated herein by reference /. Activated platelets that express GMP-140 on their surface are known to bind to monocytes and neutrophils / Jungi et al., Blood, vol. 67, pp. 629-636 / 1986 //, as well as with cell lines similar to monocytes, for example HL60 and U937 / Jungi and others, see above; Silvestein et al., J. Clin Invest, T. 79, pp. 867-874 / 1987 //; all of these works are incorporated herein by reference. GMP-140 is a membrane protein of granules of alpha with a molecular weight of 140,000, which is expressed on the surface of activated platelets by stimulating platelets and secreting granules / HSU-Lin et al., J. Biol. Chem. t. 259, p. 9121-9126 / 1984 /; Sternberg et al., J. Cell. Biol T. 101, pp. 880-886 / 1985 /; Berman et al., J. Clin. Invest vol. 78, pp. 130-137 / 1986 //. It was also found in megakaryotes / Beckstead et al., Blood, v. 67, p. 285-293 / 1986 // and in endothelial cells / Mc Ever et al., Blood v. 70, p. 355a / 1987 // inside Tel Weibel-Palad / Bonfanti et al., Blood, vol. 73, pp. 1109-1112 / 1989 //. Fury et al. In US Pat. No. 7,833,330 describe monoclonal antibodies active against GPM-140. All of the above works are incorporated herein by reference.

 The third selectin receptor is the "home" lymphocyte receptor, MEL-14 antigen or LAM-1 / Gallatin et al., Nature, T. 304, pp. 30-34 / 1983 /; Siegellman et al., Science, T. 243, pp. 1165-1172 / 1989 /; Rosen Cell Biology, vol. 1, pp. 913-919 / 1989 /; and Lasky et al., Cell, T. 56, pp. 1045-1055 / 1989 /; all of these works are incorporated herein by reference. In addition to lymphocytes, the “home” antigen MEL-14 / LAM-1 apparently functions earlier when neutrophil binds to endothelium.

 The structure and function of selectin receptors was elucidated by cloning and expression of full length cDNA encoding each of the aforementioned receptors / cm. , for example, Bevilacgua and others, Science, see above, (ELAM-1), Geng and others, see above (GMP-140), and Lasky and others, see above / MEL-14 antigen // . An extracellular portion of selectins can be divided into three segments based on homolonies with the previously described proteins. The N-terminal region (approximately 120 amino acids) is linked to the mammalian type C lectin protein family as described by Drickamer. J. Biol. Chem. T. 263, pp. 9557-9560, / 1988 / / which is incorporated herein by reference /, which includes a CD23 weak affinity IgE receptor. The following is a polypeptide segment that contains a sequence that is linked to proteins containing an epidermal growth factor (EGF) motif. Finally, after the EGF region, there are one or more tandem repeating motifs of approximately 60 amino acids each, associated with those contained in the family of additional regulatory proteins.

 Since selectin receptors contain a region resembling lectin, the specificity of these molecules is likewise based on protein-carbohydrate interactions. The facts cited here indicate that the sialylated, fucosylated N-acetylactosamine component of the Lewis X antigen, referred to herein as SLX, is a component recognized by the selectin receptor lectin zone. In particular, the analysis shows recognition of this component as ELAM-1, and GMP-140. The compounds of the present invention contain this fucosylated, sialylated N-acetylactosamine block in various configurations.

The term “selective binding,” as used herein, refers to the specific recognition by one molecule (generally referred to as a receptor) of another molecule (commonly referred to as a ligand) by spatial or polar organization of the determinant site on the second molecule. Selective binding between the two molecules takes place where the affinity is strong enough. The binding affinity generally appears to be an affinity constant (K _a ) for equilibrium concentrations of associated and dissociated configurations, namely, K _a = [RL] / [R] [L], where [R], [L] and [RL] are concentrations in the equilibrium state of the receptor [R], ligand [L] and receptor-ligand complex [RL], respectively.

 Interactions of specific binding of the receptor and ligand molecules generally include reversible non-covalent associations, such as electrostatic attraction, Van der Waals forces and hydrogen bonds. See, in general, Stryer, Biochemistry /W.H. Freeman and Company, N.Y. 3rd. Ed. 1988 /, which is incorporated herein by reference. Examples of selective binding interactions include antibody-antigen recognition, enzyme-substrate recognition, and the like.

 The nomenclature used to describe the oligosaccharide moieties in accordance with the present invention follows standard nomenclature. Standard abbreviations for individual monosaccharides are used. For example, 2-N-acetylglucosamine is indicated through GlcNAc, fucose through Fuc, galactose through Gal, and glucose through Glc. The two sialic acids that may be present on the oligosaccharides of the present invention are 5-N-acetylneuraminic acid (NeuAc) and 5-N-glycolylneuraminic acid (NeuGc). Unless otherwise indicated, all sugars except fucose (L-isomer) are D-isomers in a cyclic configuration (e.g., pyranose or furanose). Two anomers of cyclic forms are represented by α and β.

 Monosaccharides are generally joined by glycosidic bonds to form oligo- and polysaccharides. The orientation of the bond relative to the flatness of the rings is indicated by α and β. Certain carbon atoms are also noted that form a bond between the two monosaccharides. So the β-glycosidic bond between C-1 galactose and C-4 glucose is represented by Gal β 1,4 Glc. For D-sugars (for example, D-GlcNAc, D-Gal and D-NeuAc), the designation α denotes hydroxyl attached to C-1 (C-2 in NeuAc) below the plane of the ring and β above the ring. In the case of L-fucose, the designation α means hydroxyl located above the ring, and β means that it is lower.

 Once SLX has been identified as a carbohydrate ligand that mediates the adhesion of the leukocyte endothelium and platelet leukocyte cell, compounds containing SLX and related structures can be purified or synthesized de novo. As described in detail below, the present invention provides a family of compounds containing selectin binding constituents of the subject invention. For example, biomolecules can be used as carriers of this component compounds. Biomolecules, as the term is used here, include, but are not limited to, a complete list of biologically significant molecules such as amino acids (and their substitutes), oligopeptides, proteins (e.g. glycoproteins and protein hormones, fatty acids), lipids (e.g. glycolipids, phospholipids, sphingolipids and gangliosides), steroid hormones, oligosaccharides, polysaccharides and nucleic acids (e.g., deoxyribonucleic acids and ribonucleic acids). These compounds can be purified and / or synthesized in accordance with standard techniques known to those skilled in the art. In addition, the most diverse compounds bearing this moiety can be synthesized de novo, as described below.

 Once obtained, such compounds can be used for various purposes, including, for example, competing inhibition of the binding of SLX-bearing cells to cells that express selectin receptors. By linking the compounds of the present invention to cell surface selective, selectin will be prevented from interacting with the native SLX ligand on migrating cells, which interferes with the normal and pathological binding of leukocytes and other cells to endothelium or platelets. Thus, compounds that contain one or more selectin-binding constituents can serve as effective inhibitors, for example, inflammation, atherosclerosis, clot formation and other pathologies involving endothelium or platelets.

 The sialic acid residue in SLX can be in various forms, so long as the selectin binding is not significantly disturbed. In general, sialic acid is 5-N-acetylneuraminic acid (NeuAc) or 5-N-acetylneuraminic acid (NeuGc). However, other sialic acids can be used in the practice of the present invention. For a review of the various forms of sialic acid suitable for use in accordance with the present invention, see R. Schauer, Methods in Enzymology, Vol. 50, pp. 64-89 / 1987 / and Schaur, Advances in Carbohydrate Chem. Biochem, T. 40, pp. 131-234; both of these works are hereby incorporated by reference. As confirmed in Example IX below, the affinity for selectin receptors is the same if the oligosaccharide ends in NeuAc or NeuGc. Thus, the term “SLX”, as used here, refers to a minimal tetrasaccharide block (sialic acid α 1,3 Gal β 1,4 [Fuc α 1,3] GlcNAc β 1,3), in which NeuAc is sialic acid , NeuGc or other equivalent forms of sialic acid. Structures presented here that have a sialic acid residue in the form of NeuAc should be understood to include other forms, in particular, NeuGc.

The analysis below shows that a pentasaccharide containing the formula
NeuAcα2, 3 Gal β 1, 4 [Fuc α 1,3] GlcNAc β 1,3 Gal β, is a minimal structure with a significantly greater inhibitory effect than tetrasaccharide. Thus, in some preferred embodiments, the oligosaccharide will contain this pentasaccharide structure.

Other variations based on the basic SLX block are also recognized by selectin receptors. For example, the analysis in Example VIII below shows that the oligosaccharide moiety, referred to as SY2 (also known as the VIM-2 antigen), has the structure
Neu Gc α 2,3 Gal β 1, 4 Glc NAc β 1, 3 Gal β 1, 4 (Fuc α 1,3) Glc NAc β 1, 3 Gal β 1, 4 Glc β
binds selectin receptors to the same extent as SLX. The SY2 component contains two sialylated N-acetylactosamine blocks, one of which is SLX. Thus, oligosaccharides recognized by selectin receptors may contain several sialylated N-acetylactosamide blocks, at least one of which is fucosylated / see, Teimeyer et al., Proc. Natl. Acad Sci (USA), T. 88, pp. 1138-1142 / 1991 /, which is incorporated herein by reference.

 The oligosaccharide moiety of the present invention preferably ends with a sialic acid residue. In some embodiments, the implementation of the sialic acid residue can also be combined with other saccharide residues, such as a second sialic acid in the α 2,8-bond.

Alternatively, the terminal sialic acid residue can be replaced by a wide variety of radicals. Thus, some selectin binding components in accordance with the present invention have the general formula:
R ₁ - NeuAc α 2,3 Gal β 1,4 Glc NAc β 1,
in which R ₁ is R ₂ R ₃ C / CO ₂ H / -, in which R ₂ and R ₃ , which may be the same or different, are H, lower alkyl (C ₁ -C ₈ ), lower hydroxyl alkyl (C ₁ -C ₈ ), arylalkyl, alkoxyalkyl. In addition, R ₂ and R ₃ can be connected to form a 4-8 element carbocyclic or heterocyclic ring.

в которой
R является:

/Смотри, Stanley и др., J. Biol. Chem, т. 263, стр. 11374 /1988/, которая включена в качестве ссылки/.Compounds containing SLX and related structures can be obtained from cell surface glycoproteins or glycolipids from several types of cells. For example, the SLX antigen is found on N carbohydrate-linked cell surface glycoproteins of LEC11 cells, a CHO cell glycosylation mutant. LEC11 express this unique glycopeptide that contains a terminal structure that carries both sialic acid and fucose in the SLX sequence

wherein
R is:

/ See, Stanley et al., J. Biol. Chem, T. 263, p. 11374/1988 /, which is incorporated by reference.

 Using the procedure described below, it was shown that the LEC11 mutant binds to activated human vascular endothelial cells. Neither wild-type CHO cells, nor other closely related mutant glycosylation CHO cell lines without a specific glycosylation structure / SLX / showed the same level of binding.

 Other sources that can be used to produce the SLX block include any cell that naturally expresses this moiety on the carbohydrate groups of glycolipids or glycoproteins. So, polymorphonuclear neutrophils, lymphocytes, a tumor cell, or HL-60 cells were used to clear this block. Other cells that bind to activated vascular endothelium can also be used to isolate ligand / cm. , Symington et al., J. Jmmunol, T. 134, p. 2498-2506 / 1985 /, Mizoguchi et al., J. Biol Chem., T. 259, p. 11949-11957 / 1984 /, Mizoguchi and others ., J. Biol Chem., T. 259, pp. 11943-11948 / 1984 /, Paietta et al., Cancer Res. t. 48, p. 28-287 / 1988 /, which are incorporated herein by reference /.

 Compounds containing SLX or its analogues can be obtained from natural sources using techniques well known in the art for isolating surface glycopropienes, glycopeptides, oligosaccharides and glycolipids from cells / See, for example, Gerard, "Purification of glycoproteins" and Thomas et al. . "Purification of membrane proteins", both works in Guide to Protein Purification, Volume 182, Methods in Enzimology / ed. Deutscher, 1990 /, which are incorporated herein by reference. For example, LEC11 cells can be used to produce a glycoprotein or glycolipid that contains an SLX block, using, for example, the technique described by Stanley et al., Supra. In short, LEC-11 cells are infected with vascular stomatitis virus. Then, the structural carbohydrate changes induced in LEC11 are expressed on N-linked, two-whiskered, G-glycoprotein G carbohydrates. The virus is purified by equilibrium gradient centrifugation, and glycopeptides are purified using proteinase digestion as described by Stanley et al.

 In order to isolate the selectin binding component from HL-60, HT-29, colo 205, neutrophils, and lines of other cells that contain a ligand recognized by selectins, several approaches are used. Since a ligand is generally expressed on the surface of cells of these types, one approach is to isolate the plasma membrane fraction enriched in this ligand. After plasma membranes are isolated, ligands can be isolated and then identified using monoclonal antibodies, in particular those that are chemically active against SLX oligosaccharide and related structures such as monoclonal antibodies FH6, SNH3 and CSLEX-1.

In order to characterize a selectin ligand on a glycoprotein, the release of an oligosaccharide is generally the first step in a structural analysis of the oligosaccharide chain. This is achieved by chemical cleavage of the protein-carbohydrate bond or by specific release of the oligosaccharide by endoglycosidase. In most cases, different procedures can be used to establish the “right” conditions for each individual glycoprotein. Oligosaccharides bound by asparagine are released by hydrazinolysis, endoglycosidases, intensive alkaline hydrolysis and trifluoroacetolysis. O-linked carbohydrate blocks are released by alkaline β-exclusion. Oligosaccharides are separated from 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. Isolated oligosaccharides are then completely analyzed. A complete structural analysis of purified oligosaccharide blocks requires the determination of monosaccharide blocks, their ring shape, configuration / D or L /, anomeric bond / α or β /, positions of bonds between sugars and their sequence. In addition, the position of all substituent groups is established. In order to determine the positions of glycosidic bonds between monosaccharides, methylation analysis is used. The anomeric configuration of sugar residues can be determined using 500 MHz ¹ H-NMR spectroscopy. The conditions and techniques used to perform a complete structural carbohydrate analysis are described generally in the book: Beeley, Laboratory Technigues in Biochemistry and Molecycar Biology, ed. Burdon and Knippenberg, Elsevier, Amsterdam / 1985 /, which is incorporated herein by reference.

Current techniques for fully characterizing oligosaccharide sugars include the use of several analytical procedures, such as FAB-MS / fast atom bombardment mass spectroscopy /, HPAE / anion exchange chromatography at high pH / and ¹ H-NMR. These techniques are / mutually / complementary. Recent examples of how these techniques are used to fully characterize the structure of the oligosaccharide can be found in the analysis by Spellman et al., J. Biol. Chem., T. 264, p. 14100/1989 / and Stanley et al., See above. Other techniques include positive ion fast atom bombardment mass spectroscopy (FAB-MS) and gas methylation analysis by gas chromatography-electron impact mass spectroscopy / GC / EI-MS / / see EPO Patent Application No. 893055153.2, which is incorporated herein by reference as a link.

 One approach to characterizing the selectin ligand on glycolipids is to destroy cells using organic solvents, isolate glycolipids and identify glycolipids that are active against monoclonal antibodies to SLX, such as FH6, SNH3, SNH4, CSLEX-I or UTM-2, and subsequent determination of the structure of the chains of oligosaccharides. To obtain glycolipids and gangliosides that contain SLX, you can use standard techniques for obtaining glycolipids / see, for example, Ledeen et al., J. Neurochem T. 21, p. 829/1973 /, which is incorporated by reference /. For example, glycolipids are extracted from HL-60, HT-29, PMN, human leukocytes and other cell lines expressing a selectin ligand using techniques generally known to those skilled in the art / see, for example, Symington et al. , J. Jmmunol. t. 134, p. 2498/1985 / and Macher and Beckstead, Leukemia Res, t. 14, p. 119-130 / 1990 /, which are hereby incorporated by reference /. Cells are grown in suspension and harvested by centrifugation. Glycolipids are extracted from the cell tablet with chloroform / methanol 2: 1 and isopropyl alcohol / hexane / water 55:25:20, as described in Kannagi et al., J. Biol. Chem. t. 257, p. 14865/1982 /, which is hereby incorporated by reference. The resulting extracts were separated using chloroform / methanol / water / 3: 2: 1 /, Folch separation. The resulting upper extraction phase contains gangliosides, and the lower phase contains glycolipids.

 The upper phase containing gangliosides / glycosphingolipids that contain at least one component of sialic acid is isolated and separated in neutral and acidic fractions using chromatography on DEAE-Sephadex, as described in detail by Ledeen and Yu, Methods Enzymol, T. 83, p. 139/1982 /, which is hereby incorporated by reference. The resulting gangliosides were collected, lyophilized and dissolved in chloroform / methanol / 2: 1 /. The lower phase of Folch separation contains glycolipids. They are separated and isolated by preparative thin layer chromatography, using chloroform / methanol / water / 60: 35: 8 / as a solvent system, as described by Symington.

To identify these gangliosides and glycolipids that contain a selectin ligand, an immunochemical analysis of glycolipids was carried out in accordance with the procedure proposed by Magnani et al., Anal. Biochem. t. 109, p. 399/1980 /, which is incorporated herein by reference. Briefly, a portion of the ganglioside described above was subjected to thin-layer chromatography. The thin layer plate was then incubated with ¹²⁵ I-labeled CSLEX-I or another monoclonal antibody that binds specifically to SLX or similar structures. After incubation with labeled antibody, the plate was exposed to radiographic film and developed. The black dots on the x-ray film correspond to the gangliosides that bind to the monoclonal antibody, and these gangliosides were removed by scraping the corresponding areas from the silicon oxide plate and eluting the gangliosides with chloroform / methanol / water. Glycolipids are also dried and resuspended in chloroform and appear in a similar thin-layer system, and probed with a radiolabeled antibody. Structural analysis of oligosaccharides derived from glycolipids was carried out essentially as described for glycoproteins.

 Oligosaccharides containing an SLX block can be obtained from glycoproteins using techniques known in the art / see, for example. Gerard, supra, pp. 537-539 /. In general, N-glycosidase F / N-glycanase / is used to cleave N-linked oligosaccharides while O-linked groups are cleaved by endo- and acetylgalactosaminadase.

 Synthetic compounds containing SLX or its analogs, attached to various components, can be obtained depending on the specific intended application. For example, SLX can be converted into a ganglioside by binding the ceramide moiety to the C-1 reducing terminal GlcNAc block. SLX structures and related structures can also be associated with a wide variety of components, such as variously substituted amino groups, heterocyclic compounds, ether bonds with branched or unbranched hydrocarbon chains, and ether bonds with aryl or alkylaryl components. The selectin binding moiety may also be coupled to various polysaccharides, amino acids, amino acid analogs, oligopeptides or proteins, using techniques well known in the art.

 The term “alkyl”, as used here, means a branched or unbranched, saturated or unsaturated hydrocarbon chain, including lower alkyls with 1-8 carbon atoms, such as methyl, ethyl, n-propyl, butyl, n-hexyl, etc. ., cycloalkyls / 3-7 carbons, cycloalkylmethyls / 4-8 carbon atoms / and arylalkyls. The term “aryl” refers to a radical derived from an aromatic hydrocarbon by removal of one atom, for example phenyl from benzene. An aromatic hydrocarbon may have more than one unsaturated carbon ring, for example, naphthyl. The term “alkoxyl” refers to an alkyl radical attached to the remainder of the molecule with oxygen, for example ethoxyl. The term "heterocyclic compound" refers to a ring compound containing three or more atoms, in which at least one of the atoms is other than carbon (for example, N, O, S, Se, P or As). Examples of such compounds include furans, pyrimidines, purines, pyrazines, and the like. The term "oligo" refers to a polymer molecule consisting of from 2 to about 10 residues, for example, amino acids / oligopeptide /, monosaccharides / oligosaccharides / and nucleic acids / oligonucleotide /. The term "poly" refers to a polymer molecule containing more than 10 residues.

в которой
n и m, которые могут быть как одинаковыми, так и различными, являются целыми числами от 2 до 12; Y является O или S; а W является O, S или NH.For the synthesis of polyvalent forms of selectin binding constituent monomer units containing SLX or other structures can be combined to form molecules having from one to about four or more selectin binding constituents. An example of such a polyvalent form is the form in which the blocks are connected using the following components:

wherein
n and m, which may be the same or different, are integers from 2 to 12; Y is O or S; and W is O, S or NH.

в которой
Y является O или S, заместители находятся в цис- или транс-отношении.Alternatively, such a component is a 5- to 14-element ring containing two substituents, each substituent having the formula

wherein
Y is O or S, the substituents are in the cis or trans ratio.

 SLX and related structures can also be attached to various heterocyclic compounds (for example, a compound containing nitrogen atoms). In this case, the constituents are preferably bonded to the nitrogen atoms on the ring, each nitrogen being bonded to that constituent. Examples of heterocyclic compounds that are suitable for this purpose are piperazine and homopiperazine.

 Alternatively, polyvalent forms of SLX or its analogs can be created by attaching the desired component to previously obtained carrier components with several points for attachment. Examples include the attachment of SLX to the amino groups of lysine and lysine-containing peptides, proteins, glycoproteins, or aspartic side chains of such compounds.

 Another technique for preparing polyvalent selectin binding compounds is to attach the desired monosaccharide residues to polysaccharides. For example, a polysaccharide that contains a repeating block having a linear core structure SLX / i.e. without fucose residue / can be converted to a polyvalent SLX containing a polysaccharide by enzymatic fucosylation. For this purpose, type Ia, II or III native polysaccharides derived from Group B Streptococcus are used. These polysaccharides can be isolated in accordance with standard methods of sludge cells deposited in the American Type Culture Collection / Type Ia - ATCC N 12400 and N 31574; Type II - ATCC N 12973 and N 31576; and Type III - ATCC N 31577 /. See, for example, Jennings et al. Biochem, vol. 22, pp. 1258-1263 / 1983 / and Patent Publication PCT N 8706267; both of them are used here as a reference.

Стрелки в приведенных выше структурах указывают на основную цепь в молекуле каждого полисахарида. Как можно видеть, полисахарид типа Ia содержит повторяющийся блок, содержащий блоковую цепь, которая соответствует линейной структуре ядра SLX. В других двух полисахаридах основная цепь содержит структуру ядра SLX. Ферментативное фукозилирование этих полисахаридов, используя α 1,3 фукозилтрансферазу в соответствии со стандартными приемами, описанными ниже, дает поливалентное SLX-соединение. После фукозилирования повторяющиеся блоки имеют следующие формулы:

Для этой цели можно использовать целый полисахарид, а также его фрагменты. Так, можно использовать полисахариды, имеющие молекулярную массу от примерно 5000 до примерно 300000. В предпочтительном варианте молекулярная масса изменяется в области от примерно 25000 до примерно 100000. Любое число боковых цепей на полисахариде типа Ia может быть фокузилировано, чтобы полисахарид обладал активностью. В общем случае, фокузилируют от примерно 5 до примерно 200 боковых цепей, в предпочтительном варианте от примерно 50 до примерно 150.These polysaccharides contain repeating units having the formulas

The arrows in the above structures indicate the backbone in the molecule of each polysaccharide. As you can see, the type Ia polysaccharide contains a repeating block containing a block chain that corresponds to the linear structure of the SLX core. In the other two polysaccharides, the main chain contains the core structure of SLX. Enzymatic fucosylation of these polysaccharides using α 1,3 fucosyltransferase in accordance with standard techniques described below gives a polyvalent SLX compound. After fucosylation, repeating blocks have the following formulas:

For this purpose, you can use the whole polysaccharide, as well as its fragments. Thus, polysaccharides having a molecular weight of from about 5,000 to about 300,000 can be used. In a preferred embodiment, the molecular weight ranges from about 25,000 to about 100,000. Any number of side chains on type Ia polysaccharide can be focussed so that the polysaccharide has activity. In general, from about 5 to about 200 side chains are focussed, preferably from about 50 to about 150.

 The synthesis of the selectin binding component can be carried out using chemical, enzymatic or combined chemical and enzymatic strategies / see, for example, EPO Publication No. 319253, which is hereby incorporated by reference /. In a preferred embodiment (Scheme 1 below), a compound containing one or more N-acetylglucosamine blocks / GlcNAc-R / can interact sequentially with galactosyltransferase / N-acetylglucosamine β 1,4 galactosyltransferase /E.C. 2.4.1.90//, sialyltransferase / Gal β 1,4 GlcNAc α 2,3 sialyltransferase / E. C. 2.4.99.6/ or Gal β 1,3 GalNAc α 2,3 sialyltransferase / E.C. 2.4.99.4/ and fucosyltransferase / N-acetylglucosaminide α 1,3 fucosyltransferase /E.C. 2.4.1.152//, to get the final, containing SLX, structure. In this case, R may be a carrier component or an activated intermediate component that will allow attachment to the corresponding carrier component. Each enzymatic reaction uses the corresponding nucleotide sugar as a donor substrate to obtain subsequent intermediates in the synthesis of SLX. Glycosyl transfer reactions can be optimally carried out with added alkaline phosphatase (for example, from the intestines of the cow, CIAP) to "absorb" a by-product, a phosphate nucleotide that can inhibit this reaction.

Общие условия для препаративного ферментативного синтеза углеводных групп, аналогичных SLX, известны /см., например, Toone и др., Tetrahedron т. 45, стр. 5365-5422 /1989/; Wong и др., J. Amer. Chem. Soc. т. 47, стр. 5416-5418 /1982/; Unverragt и др., J. Amer. Chem. Soc. т. 112, стр. 9308-9309 /1990/; Prieels и др., J. Biol. Chem. т. 256, стр. 10456-10463 /1981/; все эти работы включены здесь в качестве ссылки/. Каждая из ключевых ферментативных реакций известна /Beyer и др., Adv. Enzymol, т. 52, стр. 23-176 /1981/; Toone и др., см. выше; и Howard и др., J. Biol. Chem. т. 262, стр. 16830-16837 /1981/; все эти работы включены здесь в качестве ссылки/. Для препаративных реакций галактозилтрансферазу и сиалилтрансферазу очищали из натуральных источников /Beyer и др., см. выше, и Weinstein и др., J. Biol. Chem. т. 257, стр. 13835-13844 /1982/, которые здесь включены в качестве ссылки/. Фукозилтрансферазы можно также идентифицировать из натуральных источников, как это в общем случае описано Grawley и Hidsgaul, Carbohyd. Res, т. 193, стр. 249-256 /1989/, которая здесь включена в качестве ссылки. Клонировали кДНК галактозилтрансферазы и сиалилтрансферазы /Paulson и Colley, J. Biol. Chem. , т. 264, стр. 17615-17618 /1989/, которая здесь включена в качестве ссылки/, допуская продуцирование растворимых рекомбинантных ферментов для крупномасштабного препаративного синтеза /Colley и др., J. Biol. Chem. т. 264, стр. 17619-17622 /1989//.

General conditions for preparative enzymatic synthesis of carbohydrate groups similar to SLX are known / see, for example, Toone et al., Tetrahedron vol. 45, pp. 5365-5422 / 1989 /; Wong et al., J. Amer. Chem. Soc. t. 47, p. 5416-5418 / 1982 /; Unverragt et al., J. Amer. Chem. Soc. T. 112, pp. 9308-9309 / 1990 /; Prieels et al., J. Biol. Chem. t. 256, p. 10456-10463 / 1981 /; all of these works are incorporated herein by reference. Each of the key enzymatic reactions is known / Beyer et al., Adv. Enzymol, T. 52, pp. 23-176 / 1981 /; Toone et al., See above; and Howard et al., J. Biol. Chem. T. 262, p. 16830-16837 / 1981 /; all of these works are incorporated herein by reference. For preparative reactions, galactosyl transferase and sialyl transferase were purified from natural sources / Beyer et al., Supra, and Weinstein et al., J. Biol. Chem. t. 257, p. 13835-13844 / 1982 /, which are hereby incorporated by reference. Fucosyl transferases can also be identified from natural sources, as is generally described by Grawley and Hidsgaul, Carbohyd. Res, t. 193, pp. 249-256 / 1989 /, which is hereby incorporated by reference. Cloned galactosyltransferase and sialyltransferase cDNAs / Paulson and Colley, J. Biol. Chem. 264, pp. 17615-17618 / 1989 /, which is incorporated herein by reference /, allowing the production of soluble recombinant enzymes for large-scale preparative synthesis / Colley et al., J. Biol. Chem. T. 264, pp. 17619-17622 / 1989 //.

 In order to obtain sufficient amounts of fucosyl transferase for large-scale reactions, the enzyme can be cloned and expressed in the form of a recombinant soluble enzyme by each person skilled in the art. As a preferred procedure, RNA can be isolated from wild-type CHO cells and LEC11 cells, as described in Chirgwin et al., Biochemistry, T. 18, pages 5214-5299 / 1979 /, and poly A + RNA can be isolated by aid chromatography on oligo / dT / cellulose. Further cDNA from LEC-11 cells can be obtained, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition / 1989 /, ed. Cold Spring Harbor Press, New York, which is hereby incorporated by reference. kDNA can be isolated using the Davis procedure / Handbook of Experimental Immunology, vol. 2, pp. 1-13 / 1986 // using excess poly A + RNA from wild-type CHO cells that do not express the target fucosyl transferase but less than they contain most of the mRNA species of LEC11 cells, the cDNA library can then be built in a CDM8 expression vector using isolated cDNA / Seed, Nature, T. 329, pp. 840-842 / 1987 //. Clones expressing fucosyl transferase can be isolated using the expression cloning procedure described in Larsen et al., Proc. Natl. Acad Sci USA, T. 86, pp. 8227-8231 / 1989 /, using transfection of COS-1 cells and sieving cells expressing the SLX antigen with a CSLEX antibody or other antibody specific for SLX antigen. A full length clone of fucosyl transferase can then be used to produce a soluble recombinant enzyme, as described by Colley et al., Supra.

Another source of SLX is glycoprotein α ₁ - acid, which is a plasma glycoprotein whose carbohydrate moieties can be fucosylated to produce SLX / cm., Alpha-Acidglycoprotein: Genetics, Biochemistry, Physiological Functions and Pharmacology, ed. Bauman et al. / Ed. Wiley 1989 /, and Walz et al., Science Vol. 250, pp. 1132-1135 / 1990 /; both of these works are hereby incorporated by reference.

 Although enzymatic or combined chemical and enzymatic synthesis of SLX compounds is preferred, chemical synthesis is also possible, as shown in Schemes II and IIa at the end of the description.

Preliminary synthesized individual "pieces" of SLX-structure. For example, the preparation of sialic acid containing glycosides, including SLX, is described in European Patent Application No. 88311312.8, which is hereby incorporated by reference. Nicolaou et al. / J. Amer. Chem. Soc., T. 112, p. 3693/1990 // described the general synthesis of the glycosine syntholipids associated with the tumor of the Le ^x family. There, the synthesis of the protected trisaccharide Gal β 1,4 / Fuc α 1,3 / GlcNAc / A / is supposed, as shown in Scheme II. The reaction of this intermediate material with an appropriate glycosyl acceptor (for example, an alcohol component) leads to compound / B /. Selective deprotection and acetylation of the glucosamine moiety was carried out essentially as described by Nicolaou et al. To obtain compound / C /. The reaction (C) with sialyl transferase as described above gives the target product SLX-P, although it can be obtained in relatively low yield using Scheme II.

 Modified fucosides can be incorporated into synthetic schemes to produce SLX analogues that vary in this moiety. For example, α-D-arabinosyl glycosides can be synthesized following known procedures, Nicolaou et al., J. Amer. Chem. Soc. T. 112, pp. 3693-3695 / 1990 / through the use of tri-O-benzyl arabinosyl halides. Other C-5 aryl or alkyl substituted arabinosyl moieties may also be synthesized, Danishefsky et al., J. Amer. Chem. Soc. t. 107, p. 1274/1985 /, Danishefsky, Aldrichimica Acta t. 19, p. 59-68 / 1986 / and the references cited therein, and introduced into the disaccharides using the same procedure. All of these works are incorporated herein by reference.

 In accordance with alternative Scheme IIa, the trisaccharide / A / is partially deprotected to obtain / D /, which is reacted sequentially with the peracetylated sialic acid methyl ester / E /, following the procedure described in Kameyama et al., XV Intl. Carbohyd. Symp Abst N A096 / 1990 / and Carbohydrate Res, t. 209, p. C1-c4 / 1991 / / which are hereby incorporated by reference /, which gives / F / after chromatographic purification. Subsequent treatment with (F) methylhydrazine, N-acetylation, O-deacetylation and hydrolysis with ester gives SLX-P.

Preferred examples of R for schemes II and IIa include alkyl (linear, branched, saturated, mono - and polyunsaturated); serine / D or L /; polypeptides containing serine; di- and tri-alkanolamines / for example, [HO / CH ₂ / _n ] ₂ NH, [HO / CH ₂ / _n ] ₃ N; where n = C ₂ -C ₂₀ in the form of a linear chain, branched, unsaturated, mono - and poly-unsaturated /. R may also be aryl substituted with aryl / for example Me, OH, I; alone or in combination comprising ¹²⁵ I /, alkylaryl, arylalkyl or other moiety which may be included by any person skilled in the art, depending on the application. The introduction of iodine into a phenolic compound, such as tyrosine, is known in the art. Radical groups containing phenols are used to introduce the radioisotope ¹²⁵ I to obtain compounds that are used in diagnostics.

 Compounds containing SLX and similar structures can also be used to analyze for the presence of other compounds that are capable of inhibiting intercellular adhesion involving selectins. Several methods can be used to analyze the biological activity of the test compounds for their ability to inhibit the reaction transmitted with the participation of selectin. Ideally, assays in accordance with the present invention allow for large-scale in vitro or in vivo screening of a wide variety of compounds.

 The agent or test compound to be screened will generally be a synthetic or naturally produced biomolecule, such as a peptide, polypeptide, protein / e.g. monoclonal antibody /, carbohydrate / e.g. oligosaccharide /, glycoconjugate, nucleic acid, etc. These compounds are synthetically produced using, for example, the oligosaccharide synthesis techniques described above (see also Khadem, Carbohydrate Chemistry / Academic Press, San Diego CA, 1988), which is incorporated herein by reference /. Various techniques for the synthesis of polypeptides of a particular composition are well known in the art / see, Atherton et al., Solid Phase Peptide Synthesis / IRL Press, Oxford 1989 /, which is incorporated herein by reference /. If the synthetic test compounds are polymeric (for example, polypeptides or polysaccharides), they are preferably modified systematically to identify a sequence of monomers that have the desired effect / see, for example, US Patent No. 4833092, which is used here as a reference /. Test compounds may also be isolated from any natural source, such as an animal, plant, fungi, or bacterial cells in accordance with standard procedures, as described above. Potentially useful monoclonal antibodies can be obtained in accordance with standard techniques described in more detail below.

 Assays in accordance with the present invention are particularly useful in identifying compounds that act as antagonists or agonists of a ligand molecule. Antagonists are compounds that reverse the physiological effect of the ligand or exclude the binding of the ligand to the receptor. The antagonist generally competes directly or indirectly with the ligand by binding to the receptor, and thus reduces the proportion of ligand molecules that bind to the receptor. In general, the antagonist will be topographically equivalent to the natural ligand and will compete directly with the ligand for the selectin binding site. Such compounds are referred to herein as "analogs" / "substitutes" /. The analogue of SLX is a molecule that structurally and functionally serves as a substitute for the SLX component in that it is recognized by the selectin receptor. Alternatively, if the ligand and the test compound can bind the receptor at the same time, this compound can act in a non-competing manner. A non-competing inhibitor acts by decreasing or inhibiting the subsequent physiological effects of the receptor-ligand interaction, and not as an agent that reduces the proportion of ligand molecules that bind to the receptor. Finally, the assays in accordance with the present invention can be used to identify synthetically or naturally occurring agonists, that is, compounds that bind a receptor and initiate a physiological response similar to a natural ligand.

 Numerous direct and indirect methods for in vitro sieving inhibitors of ligand-receptor interactions are available and are known to those skilled in the art. For example, one can determine the ability to inhibit the adhesion of SLXX-bearing cells to cells expressing specific selectin. As mentioned above, the selectin receptor genes were pre-cloned, so these genes can be inserted and expressed in a wide variety of cells, such as COS cells, CHO cells, etc. In addition, cells that usually do not express SLX, able to transform one or more glycosyltransferase genes, which give transformed cells the ability to synthesize a ligand. / See, for example, Lowe et al., Cell, t. 63, p. 475-484 / 1990 /, which is incorporated herein by reference /. In general, a test compound or agent is incubated with cells bearing labeled SLX and activated endothelial cells immobilized from a solid surface. Inhibition of cell adhesion is then determined by detecting a label associated with this surface after appropriate washing. In the assays given in the examples below, HL-80 promyelocyte cells and activated human endothelial cells or activated platelets were used.

 Since a ligand specific for selectin receptors has now been identified, isolated ligand molecules can also be used in assays. The terms “isolated selectin binding agent” or “isolated SLX moiety,” as used herein, refer to a selectin binding compound that is in a state other than its native state, for example, it is not associated with a cell membrane of a cell that normally expresses a ligand . Thus, the component of an isolated SLX may be a component of an isolated molecule, such as an oligosaccharide or glycoconjugate. An isolated molecule can be synthesized or obtained from the membranes of SLX-bearing cells. Alternatively, an isolated selectin binding agent, or SLX moiety may be associated with a liposome or attached to a solid surface before use in this assay. Techniques for preparing selectin binding liposomes and for immobilizing various biomolecules are discussed in detail below.

 In general, in vitro assays in accordance with the present invention are competing assays that detect the ability of a test compound to compete inhibition of binding of a compound that is known to bind either a receptor or a ligand. Inhibition of binding between SLX and a selectin receptor is experienced in the general case. Inhibition of other binding interactions can also be experienced, for example, inhibition of binding between a monoclonal antibody (e.g., CSLEX-I) and SLX, or between an SLX substitute and a selectin inhibitor. Numerous types of competing assays are known / see, for example, U.S. Patent Nos. 3,376,110, 4,016,043, and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Publications, N.Y. / 1988 /. which are hereby incorporated by reference.

 Assays in accordance with the present invention are also suitable for measuring the binding of a test compound to only one component, rather than using a competing assay. For example, immunoglobulins can be used to identify compounds that contain an SLX moiety. Standard procedures can be used for assaying for monoclonal antibodies, such as ELISA / cm. Harlow and Lane above. When glycolipids containing the SLX antigen are analyzed, the reactivity of the monoclonal antibody with the antigen can be analyzed by TXC immunostaining using techniques originally described in Magnani et al., Anal. Biochem. t. 109, p. 339-402 / 1980 /, or solid-state radioimmunoassay as described by Kanagi et al., Cancer Res, t. 43, p. 4997-5005 / 1983 /; which are incorporated herein by reference. Glycoproteins can be analyzed using standard immunoblot techniques, as described in Harlow and Lane, see above. Sandwich analysis formats are also suitable / see, for example, US Patent Nos. 4,642,285, 4,299,916 and 4,391,904; and Harlow and Lane, see above; all of these materials are incorporated herein by reference. In general, compounds that have been identified in a binding assay can be further tested to determine their ability to inhibit receptor-ligand interactions.

 Other assay formats include detecting the presence or absence of various physiological changes in either ligand-bearing or selectin-bearing cells that result from the interaction. Examples of such assays include measuring changes in binding transcription activity / see, for example, EPO publication N 3712820 /, detecting various extracellular effects involving cells, see, for example, PCT Publication N 90/00503 / and detecting changes in membrane potential individual cells / see, for example, US Patent N 4 343 782 /; all of these works are incorporated herein by reference. Alternatively, structural changes in isolated receptors or ligands can be detected; see, for example, US Patent No. 4,859,609, which is hereby incorporated by reference.

 Any component of the assay, including a ligand, receptor, or test compound, can be bound to a solid surface. Numerous techniques for immobilizing biomolecules on solid surfaces are known in the art. For example, the solid surface may be a membrane (for example, nitrocellulose), a microtiter cup (for example, PVC or polystyrene / or a ball. The target component may be covalently linked or non-covalently attached via non-specific binding.

 As a material for a solid surface, you can use a wide variety of organic and inorganic polymers, both natural and synthetic. Examples of such polymers include polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, poly / ethylene terphthalate /, rayon, nylon, poly / vinyl butyrate /, silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, etc. . Other materials that can be used include paper, glass, ceramics, metals, metalloids, semiconductor materials, cermets, and the like. In addition, they include materials that form gels, such as proteins, for example, gelatins, lipopolysaccharides, silicates, agarose and polyacrylamides and polymers that form several aqueous phases, such as dextrans, polyalkylene glycols / alkylene with 2-3 carbon atoms / or surface-active agents, for example, amphiphilic compounds such as phospholipids, long-chain alkyl ammonium salts (12-24 carbon atoms), and the like. So, where the solid surface is porous, various pore sizes can be used, depending on the nature of the system.

 Upon receipt of the surface, you can use a wide variety of materials, in particular laminates, to obtain various properties. For example, protein coatings such as gelatin can be used in order to avoid nonspecific binding, to simplify covalent conjugation, to enhance the detection signal, and the like.

 If it is desired to obtain covalent bonding between the compound and the surface, the surface may be generally multifunctional or capable of being multifunctional. Functional groups that may be present on the surface and used for binding may include carboxylic acids, aldehydes, amino groups, cyano groups, ethylene groups, hydroxyl groups, mercapto groups, and the like. The mechanism of binding of various compounds to various surfaces is well known and numerous illustrations are available in the literature. See, for example, Immobilized Enzymes by Ichiro Chibata Halsted Press, New York, 1978, and Cuatrecasas, J. Biol. Chem Vol. 245, p. 3059/1970 /, which are hereby incorporated by reference.

 In addition to covalent binding, various techniques can be used for non-covalent binding of the analytic component. Non-covalent binding is generally a non-specific uptake of a compound on a surface. In general, the surface is blocked by the second compound to prevent non-specific binding of the labeled assay components. Alternatively, a surface is created such that it non-specifically binds to one component, but does not substantially bind to another. For example, a lectin-bearing surface such as Concanavalin A will bind to a carbohydrate-containing compound, but not to a labeled protein that has not been glycosylated. Various hard surfaces for use in non-covalent attachment of analytical components are described in US Pat. Nos. 4,447,576 and 4,254,082, which are incorporated herein by reference.

Many assay formats use labeled assay components such as SLX ligands, SLX analogs, immunoglobulins, receptors, or test compounds. This label can be associated directly or indirectly with the target component analysis in accordance with techniques well known in the art. You can use a variety of labels. This component can be labeled with any of several known techniques. The best known detection technique is the use of autoradiography with ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C or ³² P labeled compounds, etc. The choice of a radioactive isotope depends on the preferences of the researcher in the simplicity of synthesis, variation in stability and half-life of the selected isotopes. Other non-radioactive labels include ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes and antibodies that serve as elements of a specific binding pair for the labeled ligand. The choice of label depends on the required sensitivity, ease of conjugation with the connection, stability requirements, and available equipment.

 Non-radioactive tags are often attached by indirect means. In the General case, the ligand molecule / for example, biotin / covalently linked to the molecule. The ligand further binds to an antiligand (for example, streptavidin), which is either hereditarily detectable or covalently binds to a signaling system, such as a detectable enzyme, a fluorescent compound or a chemiluminescent compound. Ligands and antiligands can be very diverse. So, where the ligand contains a natural antiligand, for example biotin, thyroxine and cortisol, it can be used in conjugation with labeled, naturally occurring antiligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.

 These molecules can be conjugated directly to signal-forming compounds, for example, by conjugation with an enzyme or fluorophore. The enzymes of interest as labels are mainly hydrolases, in particular phosphatases, esterases and glucosidases, or oxidoreductases, in particular peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansil, umbelliferone, etc. Chemiluminescent compounds include luciferin and 2,3-dihydrophthalazinedione, for example, luminol. For a review of various signal producing systems that can be used, see US Pat. No. 4,391,904, which is incorporated herein by reference.

 As discussed above, in addition to compounds of various inhibitors that contain an available SLX block or SLX analogue, the present invention also provides monoclonal antibodies capable of inhibiting intercellular adhesion involving selectins, as well as methods for identifying such antibodies. Monoclonal antibodies bind to a selectin ligand or receptor, and block cell adhesion. Thus, the most diverse techniques known to every person skilled in the art for the production and manipulation of molecules of various immunoglobulins can be used to inhibit intercellular adhesion.

The term "immunoglobulin," as used here, refers to a prototype consisting of one or more polypeptides that are essentially encoded by immunoglobulin genes. The genes of the “recognizable” immunoglobulin include the genes for the constant zones of kappa, lambda, alpha, gamma, delta, epsilon and mu, as well as myriad of genes for the variable zones of immunoglobulin. Immunoglobulins can exist in a wide variety of forms besides antibodies, including, for example, Fv, FAB and F (ab) ₂ , as well as single chains / for example, Huston et al., Proc. Nat. Acad Sci. USA, T. 85, pp. 5879-5883 / 1988 / and Bird et al., Science, T. 242, pp. 423 - 426/1988 /, which are incorporated herein by reference /. / see, in the General case, Hood and others, Immunology, 2nd ed., ed. Benjamin, NY / 1984 / and Hunkapiller and Hood Nature vol. 323, p. 15-16 / 1986 /, which are hereby incorporated by reference.

 Antibodies that bind the SLX antigen can be produced by a wide variety of agents. The production of non-human monoclonal antibodies, for example, murine, lagomorpha, equine, etc. it is well known and can be carried out by, for example, immunizing an animal with a SLX antigen or a preparation containing a glycoprotein or glycolipid containing an antigen. Antibody-producing cells derived from immunized animals were isolated and sieved or first sieved to produce an antibody that inhibits the interaction of the viral surface protein with the receptor molecule, and then isolated. For a discussion of the general procedure for producing monoclonal antibodies, see Harlow and Lane, Antibodies, A Laboratory Manual / 1988 /, see above.

The formation of human monoclonal antibodies relative to the human antigen / in the case of an SLX block isolated from human tissue / may be difficult using known techniques. Therefore, it may be desirable to transfer the binding zones of the antigen of non-human antibodies, for example, F / ab '/ ₂ or hypervariable zones to human constant zones / Fc /, or equipped zones using recombinant DNA techniques to produce essentially human molecules. Such techniques are generally known in the art and are described, for example, in U.S. Patent Nos. 4,816,397, EP Publications Nos. 173,494 and 239,400, which are incorporated herein by reference. Alternatively, DNA sequences that induce a human monoclonal antibody or fractions thereof that specifically binds to human SLX can be isolated by sifting a DNA library from human B cells in accordance with the general description summarized in Huse et al., Science T. 246, pp. 1275 - 1281 (1989), which is hereby incorporated by reference, and then the cloning and amplification of sequences that encode an antibody / or binding fragment / with target specificity.

Several currently available monoclonal antibodies can be used in accordance with the present invention in order to inhibit intercellular adhesion involving selectins. For example, CSLEX-I / cm., Campbell et al., J. Biol. Chem. T. 259, p. 11208 - 11214/1984 //, YIM-2, which recognizes a sequence slightly different from SLX / cm, Macher et al., see above /, FH6 / described in US patent N 4 904 596 / / all of these works are incorporated herein by reference / or SH ₃ and SH ₄ obtained by Dr. S. Hakomori from the Biomembran Institute in Seattle, Washington.

 The compounds of the present invention, including immunoglobulins, can be used in the preparation of pharmaceutical forms, as discussed in detail below. If the compound is an oligosaccharide or glycoconjugate, the SLX or SLX substitute moiety can be represented in a wide variety of forms, but it must be able to efficiently bind to a selectin receptor, such as ELAM-1, GMP-140 or MEL-14 antigen, while inhibiting intercellular adhesion.

 The pharmaceutical compositions of the present invention can be used to block or inhibit cell adhesion associated with several of the diseases. For example, several inflammatory diseases are associated with selectins expressed on vascular endothelial cells and platelets. The term "inflammation" is used here to refer to the reactions of both, specific and non-specific, defense systems. The reaction of a specific defense system is the specific response of the immune system to an antigen. An example of specific reactions of the defense system includes the reaction of an antibody to antigens such as viruses and delayed-type hypersensitivity. A non-specific reaction of the defense system is an inflammatory reaction transmitted by white blood cells, which are generally devoid of immunological memory. Such cells include macrophages, eosinophils and neutrophils. Examples of non-specific reactions include immediate bloating after a bite and the accumulation of PMN leukocytes at the sites of bacterial infections (for example, pulmonary infiltrates with bacterial pneumonia and the formation of pus with abscesses).

 Other disorders that can be treated in accordance with the present invention include rheumatoid arthritis, post-ischemic tissue damage involving white blood cells / disorders with repeated perfusion /, lesions or shock during frostbite, acute / with white blood cells / lung disease / e.g. respiratory distress syndrome in adults /, asthma, traumatic shock, septic shock, nephritis and acute and chronic inflammation, including atopic dermatitis, psoriasis and inflammatory bowel disease. In accordance with the present invention, various platelet pathologies, such as atherosclerosis and clot formation, can also be treated. In addition, the formation of tumor metastases can be inhibited or prevented by inhibiting the adhesion of circulating cancer cells. Examples include colon carcinoma and melanoma.

 Using the example, it was found that lesions resulting from repeated perfusion are particularly susceptible to treatment with the compositions of the present invention. Compositions that inhibit the selectin GMP-140 ligand interaction may be particularly useful in the treatment or prevention of lesions during repeated perfusion. The present invention can be used prophylactically before heart surgery to accelerate post-surgical recovery.

 Due to the fact that GMP-140 is stored in the Weibel-Palada bodies of platelets and endothelial cells, and is released upon activation by thrombin to participate in the adhesion of neutrophils and monocytes, inhibitors of the interaction with GMP-140 ligand can be especially effective in minimizing tissue lesions that often accompanied by thrombotic disorders. For example, such inhibitors can be of therapeutic value for patients who have recently suffered a stroke, myocardial infarction, deep vein thrombosis, pulmonary embolism, etc. These compounds are especially useful in prophylactic therapy.

 The compositions of the present invention find, in particular, the use in the treatment of secondary effects of septic shock or disseminated intravascular coagulation / DIC /. Migration of leukocytes into the tissue during septic shock or DIC often leads to pathological tissue destruction. In addition, these patients may have widespread inflammation from the microcirculating thrombus and diffusion. The therapeutic compositions of the present invention inhibit the migration of white blood cells to these places and mitigate tissue damage.

 Selectin-ligand interaction inhibitors are also useful in the treatment of traumatic shock and acute tissue lesions that are associated with it. Due to the fact that selectins play the role of replenishing white blood cells at the lesion sites, in particular, ELAM-I in cases of acute lesions and inflammations, their inhibitors can be applied locally or systematically to control tissue damage associated with such lesions. In addition, due to the specificity of such inhibitors for inflammation sites, for example, where ELAM-I receptors are expressed, these compositions will be more effective and unlikely to cause complications when compared with known anti-inflammatory agents.

 Thus, in accordance with the present invention also provides pharmaceutical compositions that can be used in the treatment of the aforementioned diseases. These pharmaceutical compositions consist of biomolecules or other compounds that contain an SLX block, antibodies that bind to SLX, or other compounds that inhibit the interaction between the SLX ligand and selectin receptors, together with pharmaceutically acceptable carriers. The biomolecule in accordance with the present invention may be a peptide, polypeptide, protein / eg immunoglobulin /, carbohydrate / e.g. oligosaccharide or polysaccharide /, glycoconjugate / e.g. glycolipid or glycoprotein /, nucleic acid, etc. These pharmaceutical compositions are suitable for use in a wide variety of drug delivery systems. For a brief overview of current methods for producing drug delivery vehicles, see Langer, Science Vol. 249, pp. 1527-1533 / 1990 /, which is incorporated herein by reference.

 Due to the complexity of the inflammatory response in mammals, anyone skilled in the art should understand that the pharmaceutical compositions of the present invention may contain SLX-bearing compounds in admixture with other compounds that are known to interfere with the function of other cell adhesion molecules. For example, members of the integrin family of adhesion molecules apparently play a role in leukocyte extravasation at the sites of infection. For a review of intercellular adhesion receptors, including selectin receptors, and their role in immune function, see Springer, Nature, T. 3463, pp. 425-434 / 1990 /, which is incorporated herein by reference. In addition, sequential treatment using pharmaceutical compositions of the invention may also be determined by the state of development of the disease to be treated. Since different adhesion molecules can be adjusted from above or below in terms of response to various factors during a disease or symptoms, one skilled in the art should know that different pharmaceutical compositions may be required to treat various inflammatory conditions.

 In one embodiment, the SLX ligand pharmaceutical composition can be used to "target" known anti-inflammatory drugs or other agents to specific tissue lesion sites. By using a selectin-binding component, such as an SLX ligand or SLX substitute, to "target" a drug to a selectin receptor, for example, on vascular endothelial cells, higher concentrations of such drugs at the lesion sites can be achieved. Side effects from the use of known anti-inflammatory chemotherapeutic agents can be substantially alleviated by using lower doses, localizing the agent at the sites of damage and / or encapsulating the agent before delivery.

 The “target” component, i.e. The SLX ligand or SLX substitute that binds to the target selectin can be directly or indirectly "paired" with a chemotherapeutic agent. Mating, which can be accomplished using means generally known in the art, should not substantially inhibit the ability of the ligand to bind to the receptor and should not substantially reduce the activity of the chemotherapeutic agent. A wide variety of chemotherapeutic agents can be paired to "target". For example, anti-inflammatory agents that can be paired include SLX-bearing compounds of the invention, immunomodulators, platelet activating factor antagonists (PAF), cyclooxygenase inhibitors, lipoxygenase inhibitors and leukotriene antagonists. Some preferred constituents include cyclosporin A, indomethacin, naproxen, FK-506, mycophenolic acid, etc. Similarly, antioxidants, such as superoxide dismutase, are used to treat lesions during repeated perfusions when the “targeting” is carried out using an SLX ligand or substitute. Similarly, anti-cancer agents can be targeted by mating the SLX ligand or as a substitute with a chemotherapeutic agent. Examples of agents that can be paired include daunomycin, doxorubicin, vinblastine, bleomycin, etc.

 The "targeting" of the selectin receptor can also be carried out via dual pathways or molecules of a dual nature (polar: nonpolar), which exist as aggregates in an aqueous solution. These dual pathways include non-polar lipids, polar lipids, mono- and diglycerides, sulfatides, lysolecithin, phospholipids, seponin, bile acids and salts. These molecules can exist as emulsions and foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions and lamellar layers. They are here generally referred to as liposomes. In these preparations, the drug to be delivered is included as part of the liposome along with the SLX ligand or substitute that binds to the selectin receptor. Thus, liposomes filled with the necessary chemotherapeutic agent can be directed to the site of tissue damage using the selectin-SLX ligand interaction. When liposomes reach areas close to damaged cells, they release selected therapeutic compositions.

 The liposomes of the present invention are formed from standard carrier-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol such as cholesterol. The choice of lipids is generally dictated by factors such as liposome size and liposome stability in the blood stream.

 In general, the main component of a lipid in liposomes is phosphatidylcholine. Phosphatidylcholines containing the most diverse groups of acyl chains with different chain lengths and degrees of saturation are available or can be isolated or synthesized using well-known techniques. In general, more saturated phosphatidylcholines are more easily dimensioned, in particular, liposome salts should be smaller than about 0.3 microns for filter sterilization purposes. The techniques used to impart the required liposome size and filter sterilization properties are described below. The composition of the acyl chains of the phospholipid can also violate the stability of liposomes in the blood. One preferred phosphatidylcholine is partially hydrogenated egg phosphatidylcholine.

 Targeting liposomes using a wide variety of targeting agents (for example, ligands, receptors, and monoclonal antibodies) is well known in the art / see, for example, US Patent Nos. 4,957,773 and 4,603,044, which are incorporated herein by reference. links. As the "targeting" agents, glycoproteins and glycolipids with a wide variety of molecular weights can be used. In general, glycoproteins are used having a molecular weight of less than about 300,000 daltons, preferably from about 40,000 to about 250,000, more preferably from about 75,000 to about 150,000. Glycolipids with a molecular weight of less than about 10,000 daltons are used, in a preferred embodiment, from about 600 to about 4000 daltons.

 Standard techniques can be used to mate targeting agents with liposomes. These techniques generally include introducing into the liposomes liquid components, such as phosphatidylethanolamine, which can be activated to attach targeting agents or the resulting lipophilic compounds, such as the lipid from which bleomycin is derived. Antibody-targeted liposomes can be constructed using, for example, liposomes that contain protein A / cm., Renneisen et al., J. Biol. Chem. T. 65, pp. 16337-16342 / 1990 / and Leonetti et al., Proc. Natl. Acad Sci. USA vol. 87, p. 2448 - 2451/1990 /, which are hereby incorporated by reference.

 Aiming mechanisms generally require that the “targeting” agents be placed on the surface of the liposome so that the “target” agents are available for interaction with the selectin receptor. A liposome is generally designed in such a way that a portion of the connecting agent is first incorporated into the membrane during the formation of the membrane. Part of the connecting means should have a lipophilic part, which is firmly embedded and fixed in the membrane. It should also have a hydrophilic moiety, which is chemically accessible on the water surface of the liposome. The hydrophilic moiety is selected so that it is chemically suitable to form a stable chemical bond with the targeting agent, which is added later. Thus, the molecule of the connecting agent / connector / must have both a lipophilic attachment and a hydrophilic chemically active group suitable for interacting with the target agent and keep the target agent in its correct position, starting from the surface of the liposome. In some cases, it is possible to attach the agent gel directly to the connector molecule, but in most cases it is more convenient to use a third molecule, which acts as a chemical bridge, which thus binds the molecule of the connector, which is located in the membrane, to the target agent, which protrudes in three-dimensional space on the surface of the bubble.

 Liposome charge is an important determinant in the purification of blood liposomes, and negatively charged liposomes are selected faster using the reticuloendothelial system / Juliano, Biochem. Biophys. Res. Commun. t. 63, p. 651/1975 // and, thus, have a shorter half-life in the blood stream. Longer circulating liposomes are generally desirable for therapeutic or diagnostic use. Liposomes that can be maintained for 8, 12, or up to 24 hours in the blood stream provide sustained release of the selectin ligand inhibitors of the invention, or can facilitate the targeting of inhibitors (which can be labeled to provide an in vivo diagnostic view) target locations before they are removed using the reticuloendothelial system.

 In general, liposomes are prepared with about 5-15 molar percent of negatively charged phospholipids, such as phosphatidylglycerol, phosphatidylserine or phosphatidylinositol. Added negatively charged phospholipids, such as phosphatidylglycerol, also serve to prevent spontaneous aggregation of liposomes and thus minimize the risk of undesired liposome aggregation. Membrane curing agents, such as sphingomyelin and saturated neutral phospholipid, at a concentration of at least about 50 molar percent and 5-15 molar percent of monosialylganglioside can provide higher circulation of the liposome preparation in the blood stream, as is generally described in US Pat. No. 4,837,028, which included here as a reference.

 In addition, the liposome suspension may include lipid protecting agents that protect the lipids and components of the drug against damage by free radicals and as a result of fat oxidation during storage. Preferred are lipophilic free radical terminating agents, such as alfatocopherol and water soluble iron specific compounds, chelating compounds such as ferrioxyanin.

 A wide variety of techniques can be used to prepare liposomes, as described, for example, in Szoka et al., Ann. Rev. Biophys. Bioeng T. 9, p. 467/1980 /, U.S. Patent Nos. 4,235,871, 4,501,728 and 4,837,028, which are incorporated herein by reference. One such technique makes it possible to obtain multilayer bubbles of heterogeneous sizes. In this technique, the lipid-forming lipids are dissolved in an appropriate organic solvent or solvent system and dried under vacuum or inert gas to form a thin lipid film. If desired, the film can be redissolved in an appropriate solvent, such as tertiary butanol, and then lyophilized to obtain a more homogeneous lipid mixture, which is in a more easily hydratable, powder-like form. This film was coated with an aqueous solution of a “targeted” drug and a “targeting” component and allowed to hydrate in the general case for 15-60 minutes with stirring. The size distributions of the resulting multilayer vesicles can be shifted in the direction of smaller sizes by hydrating lipids under conditions of more vigorous stirring or by adding solubilizing detergents such as deoxycholate.

 The hydration medium contains the target drug at a concentration that is desired in the internal volume of the liposomes in the final suspension of liposomes. In general, a drug solution contains 10-100 mg / ml in buffered saline. The concentration of the targeting SLX molecule or substitute that binds to selectin is generally about 0.1-20 mg / ml.

 Following the procedure for obtaining liposomes, liposomes can have the required size range and a 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 known filter, generally a 0.22 micron filter. The filter sterilization procedure can be carried out on the basis of high permeability, if liposomes should have a size of about 0.2-0.4 microns.

 There are several techniques that provide liposomes of the required size. One such method of providing a predetermined size is described in US Pat. No. 4,737,323, which is hereby incorporated by reference. The destruction of the suspension of liposomes by ultrasound either in the bath or using an ultrasound probe gives a gradual reduction in size to small single-layer vesicles with a size of less than about 0.05 microns. Homogenization is another technique that relies on shear energy to split large liposomes into smaller liposomes. In the general homogenization procedure, the multilayer vesicles were recycled through a standard emulsion homogenizer until the selected liposome sizes were obtained, generally from about 0.1 to 0.5 microns. In both procedures, the particle size distribution can be analyzed using a known particle size separation using a laser beam.

 Extrusion of liposomes through a small pore polycarbonate membrane or asymmetric ceramic membranes is also an effective technique for reducing liposome size to a relatively good size distribution. In general, the suspension passes through the membrane one or more times until a target size distribution of liposomes is obtained. Liposomes can be extruded through membranes with successively decreasing pore sizes in order to achieve a gradual reduction in liposome size.

 Even with the most effective encapsulation procedures, a liposome suspension in size at the initial moment may contain up to about 50% or more of the drug and the targeting agent in free / not included in the capsule / form. Therefore, in order to optimize the benefits of a liposomal “targeted” drug, it is important to remove the free drug and the targeting agent from the final injectable suspension.

 There are several techniques that can be used to remove an unreached compound from a liposome suspension. According to one of these techniques, the liposomes in suspension are converted into tablets by high speed centrifugation, which transfers the free compound and very small liposomes to the top layer. Another such technique involves concentrating the suspension by ultra-filtration, followed by suspending the concentrated liposomes in a drug-free medium. Alternatively, gel filtration can be used to separate large liposome particles from soluble molecules.

 After treatment in order to remove the free drug and / or the “targeting” agent, the liposome suspension is adjusted to the target concentration for internal use. This may include re-suspension of the liposomes in an appropriate volume of the injectable medium, where the liposomes were concentrated, for example, by centrifugation or ultrafiltration, or concentration of the suspension, where the stage of removal of the drug increased the total volume of the suspension. The suspension was then sterilized by filtration as described above. The preparation of the liposome ligand can be used parenterally or topically in a dose that varies, for example, depending on the method of application, the drug delivered and the type of disease to be treated, etc.

 For pharmaceutical compositions that contain an SLX ligand and / or SLX substitute that bind to selectin receptors, the dose of this compound will vary depending, for example, on the particular compound, method of use, the particular disease to be treated, and its severity, general the condition and health of the patient and the instructions of the attending physician. For example, to treat injuries caused by repeated perfusion, the dose varies from about 50 μg to 2000 mg / day for a patient weighing 70 kg. Ideally, therapeutic use should begin as quickly as possible after myocardial infarction or other injuries. These pharmaceutical compositions are intended for parenteral, topical, dental or local use, such as aerosols or through the skin, for prophylactic and / or therapeutic treatment. The pharmaceutical compositions can be applied in a wide variety of unit dosage forms, depending on the mode of administration. For example, unit dosage forms intended for dental use include powders, tablets, pills, capsules, and dragees.

 In a preferred embodiment, the pharmaceutical compositions are administered intravenously. Thus, in accordance with the present invention, there are provided compositions for intravenous administration which comprise a solution of a compound dissolved or suspended in a pharmaceutically acceptable carrier, preferably in an aqueous carrier. A wide variety of carriers can be used, for example, water, buffered water, 0.4% saline, and the like. These compositions can be sterilized using well-known sterilization techniques, or they can be sterilized on a filter. The resulting aqueous solutions can be packaged for use or they can be lyophilized, the lyophilized preparations being combined with a sterile aqueous solution before use. These compositions may contain pharmaceutically acceptable auxiliary materials that are required to approximate physiological conditions, such as pH regulators and buffering agents, tonicity 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 the SLX ligand or substitute that can be combined with other SLX ligands or substitutes to form a “cocktail” to increase the effectiveness of the pharmaceutical form can vary widely, namely, from less than about 0.05%, in general not less than about 1% to 10-30 mass%, and it can be selected mainly on the basis of the volume of liquid, viscosities, etc., in accordance with a particular application. Such a cocktail may contain a monoclonal antibody that binds to a selectin receptor, for example, an ELAM-I or GMP-140 monoclonal antibody coupled to an SLX ligand, a ligand substitute or a monoclonal antibody to this ligand so as to effectively inhibit the ligand-receptor interaction . As described above, the components of the cocktail can be delivered using liposome preparations.

 Thus, a general pharmaceutical composition for intravenous infusion may contain 250 ml of sterile Ringer's solution and 25 mg of the compound. Real examples for the preparation of parenterally applicable compounds are known or obvious to every person skilled in the art and are described in more detail, for example, in Remington's Pharmaceutical Sciences 17th edition, ed. Mack Publishing Company, Easton PA / 1985 /, which is incorporated herein by reference.

 For solid compositions, known non-toxic solid carriers can be used, which include, for example, pharmaceutical varieties of mannitol, lectose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. For dental use, a pharmaceutically acceptable non-toxic composition is formed by the administration of any of the known excipients, such as those carriers listed above, and generally 10-95% of the active ingredient, i.e. one or more SLX ligands or substitutes which are the subject of the present invention, in a preferred embodiment, about 20%, (see Remington's, above).

 For aerosol use, the compounds are preferably used in finely divided form together with a surface-active agent and a spraying agent. The total percentage of SLX-oligosaccharide ligand or substitute is 0.05-30 mass%, in the preferred embodiment, 1-10%. The surface active agent should, of course, be non-toxic and, preferably, soluble in the nebulizing agent. Representatives of such agents are 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 aliphatic polyhydric alcohol or its cyclic anhydride, such as, for example, ethylene glycol, glycerin, erythritol, arabitol, mannitol, sorbitol, hexite anhydrides derived from sorbitol, and derivatives of the polyoxyethylene and polyoxypropylene of these esters. Mixed esters, such as mixed or natural glycerides, may be used. The surface active agent may comprise 0.1-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is generally a spray agent. Liquefied spraying agents are generally gases under ambient conditions and condense under pressure. Among the corresponding liquefied atomizing agents are lower alkanes containing up to 5 carbon atoms, such as butane and propane; and in a preferred embodiment, fluorinated or fluorochlorinated alkanes. Mixtures of the above materials may also be used. Upon receipt of the aerosol, a container equipped with an appropriate valve is filled with an appropriate nebulizing agent containing finely divided compounds and a surface-active agent. The ingredients are thus maintained at elevated pressure until they are released by the valve.

 Compositions containing these compounds can be used by prophylactic and / or therapeutic administration. In therapeutic applications, the compositions are applied to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially stop the symptoms of the disease and its complications. An amount adequate to achieve this goal is defined as a “therapeutically effective dose”. Amounts effective for such use will depend on the severity of the disease, weight and general condition of the patient, but generally varies from about 0.5 mg to about 2,000 mg of SLX oligosaccharide or SLX substitute per day for a patient weighing 70 kg with a dosage of from about 5 mg to about 200 mg of the compound per day in a preferred embodiment.

 In prophylactic applications, compositions containing the compounds of the present invention are applied to a patient susceptible to, or in other words, at risk of a particular disease. This amount is defined as a "prophylactically effective dose." In such an application, the exact amounts again depend on the condition of the patient and his weight, but generally varies from about 0.5 mg to about 1,000 mg per patient weighing 70 kilograms, more preferably from about 5 mg to about 200 mg for a patient weighing 70 kg.

 A single or multiple use of the compositions can be carried out with dose levels and frequency as chosen by the attending physician. In any case, the pharmaceutical form should provide an amount of the SLX oligosaccharide or SLX substitute that is the subject of the present invention, sufficient for effective treatment of the patient.

These compounds may also find use as diagnostic reagents. For example, labeled compounds can be used to localize areas of inflammation or tumor metastasis in a patient who is suspected of having an inflammatory disease. For such use, the compounds can be “labeled” with ¹²⁵ I, ¹⁴ C or tritium.

 The following examples serve the purpose of illustration and should not be construed as limiting the present invention.

 Example I. Isolation of α 1,3, -fucosyltransferase 1 from the Golgi apparatus.

 LEC11, HL-60, HT-29, some adenocarcinomas (in particular, colo 205 cells) and polymorphonuclear leukocytes / PMN, neutrophils / contain very specific α 1,3-fucosyltransferase 1, which is able to transfer fucose from GDP-fucose to sialylated substrates Neu Ac α 2,3Gal β 1,4GlcNAc or NeuGc α 2,3Gal β 1,4GlcNAc.

 It is well known in the art that fucose is transferred to oligosaccharide chains in the cavity of the Golgi apparatus through specific fucosyltransferases, a review of which is contained in Schacter and Roseman's book "The Biochemistry of Hycoproteins and Proteoglycans" ed. W. Lennary ed. Plenum Press, New York, pp. 85-160 / 1980 /, which is incorporated herein by reference. Since the subcellular localization of fucosyl transferase is located in the Golgi apparatus, the first step in isolating these enzymes is to isolate a fraction of the Golgi apparatus from a cell line that expresses this new and specific α 1,3-fucosyl transferase.

The bubble fractions obtained in the Golgi apparatus were treated with the modified procedure described in Balch et al., Cell, vol. 39, p. 405/1984 /, which is hereby incorporated by reference. LEC11, HL-60, HT-29, PMN, colo 205, and other cell lines containing α 1,3-fucosyltransferase 1 were grown in suspension to a density of approximately 5 • 10 ⁵ cells / ml. Cells were collected from suspension culture by centrifugation at a speed of 2000 x g. The resulting cell tablet from 12 liters of suspension / 6 • 10 ⁹ cells / was again suspended in 3 volumes / packed cell volume / ice-cold 0.25 M sucrose solution / m / o / containing Tris-Cl / 10 mM /, pH 7 , 0, heat-deactivated cow germ serum (7%) and Aprotinin / 100 μg / ml, Sigma Chemical, Co., St. Louis, Mo./.

Cells were destroyed (with approximately 60 strokes) using a tight-fitting Wheaton glass homogenizer and pestle A. The homogenate was centrifuged for 5 min at a speed of 500 x g using a benchtop clinical centrifuge. The lipid and insoluble material remaining in the upper part of the solution in a centrifuge tube was discarded. The turbid upper layer was transferred to a clean tube, and then the sucrose concentration in the upper layer fraction was ensured at the level of 40% / m / o / sucrose in Tris-Cl / 20 mM /, pH 7.0, using a refractometer. Five milliliters of this suspension was transferred to an ultracentrifugation tube and successively applied with a layer of 2.5 ml of 35% / m / o / sucrose in Tris-Cl / 10 mM, pH 7.0 / and 2.0 ml 29% / m / o / sucrose in 10 mM Tris-Cl buffer. The centrifugation gradient was 1 hour at a speed of 110,000 x g using a SW-41 / Beckman / engine at a temperature of 5 ^° C. Bubbles enriched with a Golgi apparatus were collected using a boundary phase of 29-35% sucrose. Other subcellular fractions were found in other boundary phases of the gradient, for example, vesicles obtained from a strip of coarse and smooth endoplasmic reticular tissue, below the vesicles obtained using the Golgi apparatus, etc. The band extracted from the boundary phase of 29-35% was analyzed for the presence and amount of sialyltransferase activity.

 The only thing known about the sialyltransferase enzyme is that it is inside the vesicles obtained from the Golgi apparatus, and that it is used by specialists in this field of technology as a marker to ensure the authenticity of the band collected from the boundary phase of 29-35%.

 Sialyltransferase assays were carried out using azialofetuin as an acceptor, as described in Briles et al., J. Biol. Chem., T. 252, p. 1107/1977 /. A good preparation of vesicles obtained on a Golgi apparatus from LEC cells generally has a specific sialyltransferase activity of 3.0 nmol / mg protein / hour.

 The resulting preparation on a Golgi apparatus was then used as a source of α 1,3-fucosyltransferase 1 used in the enzymatic synthesis described above.

 Example II Evidence of intercellular adhesion using cells expressing SLX.

The ability of LEC11 cells / that express SLX / to bind to activated endothelial cells expressing ELAM-I was compared with the same ability of CHO cells and another glycosylation mutant, LEC12, which expresses the Le ^x structure, a non-sialylated form of SLX.

 Materials

 Passage 5 of the endothelial cells of the human umbilical vein / HUYEC / / Clonetics /, which was grown on a gelatin-coated tablet with 48 recesses, was used as a source of endothelial cells. Cells were stimulated with IL-1 β / Genzyme / at a concentration of 30 μg / ml. Cells were stimulated exactly for 4 hours. HL-60 cells obtained from the American Type Culture Collection / ATCC N CCL 240 / were used as a source of control ligand-bearing cells. They were collected from the culture volume in RPM1 1640 / Gibco / containing Penicillin / 100 units / ml / / Streptomycin / 100 μg / ml / / Irvine Scientific /, L-glutamine / 2 mm / / Irvine Scientific / and 10% Serum fetal cow / company Hazleton / / hereinafter referred to as CPPMI /. LEC11, LEC12 and CHO-KI are represented by Dr. Stanley / P. Stanley /. They were grown in suspension culture in complete MEM alpha medium containing ribonucleotides and deoxyribonucleotides / Gibco /, Penicillin / 100 units / ml / / Streptomycin / 100 μg / ml / / Irvine Scientific /, L-Glutamine / 2 mm / / company Irvine Scientific /, L-Glutamine / 2 mM / / Jrvine Scientific / and 10% Cow Germ Serum / Hazelton /.

 Procedure.

1. HL-60, LEC11, LEC12, and CHO-K1 cells were harvested and washed in CRPMI. Counting live cells was done using trypan blue. From 3 • 10 ⁶ cells of each type, tablets were made in a 10 ml test tube and 300 μl ⁵¹ Cr / 450 μCi / / New England Nuclear / was added to each tablet. The tubes were incubated for 1 hour at 37 ^o C with gentle stirring.

2. The labeled cells were washed 3X in medium, and resuspended in 2 • 10 ^5/400 .mu.l / ml 6 /. Then the tubes were placed in an ice bath at a temperature of 4 ^o C.

3. After 4 hours of incubation with IL-1β, the analytical plate containing activated HOYEK was removed from the incubator, and this process was quickly stopped for 15 minutes, the plate was kept in an ice bath at 4 ^o C.

 4. When the temperature in both samples equalized, the medium was removed from the recesses with a Pasteur pipette, from several recesses at the same time.

5. Labeled cells were added to the wells in a volume of 400 μl, equivalent to 2 • 10 ⁵ cells / well. Three portions of 400 μl of each cell suspension were placed in glass tubes to determine the PMR / number of samples / minute /.

 6. The plate was incubated in an ice / water bath for 30 minutes.

 7. Unbound cells were removed from the recesses of the assay plate by systematic resuspension using a Pasteur pipette, then by adding and removing 0.7 ml of medium.

8. The whole medium was removed from the recesses and a solution of 0.125 M Tris, 2% SDS / sodium dodecyl sulfate / and 10% glycerol / 0.3 ml / was added. The plate was held for 30 minutes, then 0.5 ml of dH ₂ O was added to each well.

 9. The fluid in each well was resuspended using a P1000 pipette and transferred to a glass tube. The P1000 tip was inserted into a test tube.

 10. Tubes, including those that contain input FOM samples / FOM — number of samples / minute /, were analyzed on a gamma counter.

 11. The FOM associated with each well was divided by the input FOM for each sample to determine binding in%. Triple analysis points for the mean and standard deviation were plotted on the coordinates.

The results obtained in this experiment are shown in FIG. 1 indicate that cells expressing SLX have the ability to bind efficiently to activated vascular endothelial cells expressing ELAM-1. These data show that LEC11 cells that express high concentrations of a single SLX carbohydrate bind exceptionally well to those activated with 1L-1 β HUYEC, while LEC12 and CHO-K1, which do not produce significant amounts of this carbohydrate, are weak binding agents of activated HUYEC. This conclusion, in addition, is confirmed by the observation that such binding takes place at a temperature of 4 ^o C, specific for binding with the participation of ELAM-1.

 Example III Inhibition of intercellular adhesion using monoclonal antibodies specific for SLX.

 Two series of experiments have been described which confirm that the neutrophil ligand for ELAM-1 contains an oligosaccharide in which the terminal sugars are Neu Ac α 2,3 Gal β 1,4 / Fuc α 1,3 / Glc NAc / SLX /.

These experiments were performed by analyzing the ability of monoclonal antibodies specific for sialylated Le ^x and non-sialylated Le ^x to block adhesion involving ELAM-1 HL-60 cells stimulated with 1L-1 β HUYEC.

 A. Lane 1 monoclonal antibody.

 Materials: HUYEC passage 3, from cultures initiated for the present experiments, was used as described above. Two series of triple depressions were left unstimulated as controls. Four triple depressions were stimulated with 1L-1 β / Genzyme / at a concentration of 10 μg / ml and 4 at a concentration of 20 μg / ml. Cells were stimulated exactly 4 hours. HL-60 cells obtained from the American Type Culture Collection (ATCC) were used as a source of ligand-bearing cells. They were collected from the culture volume in RPMI-1640 / Gibco / containing penicillin / 100 units / ml /, streptomycin / 100 μg / ml / / Irvine Scientific, L-Glutamine / 2 mm / / Irvine Scientific / and 10% serum fetal cow / company Hazleton / / hereinafter referred to as cRPM1 /.

 Monoclonal antibody preparations included SNH3 / IgM / at a concentration of about 20 μg / ml and SH1 / IgG3 / at a concentration of about 10 μg / ml. The specificity of SNH3 is directed towards SLX while SH1 “recognizes” the non-sialylated structure.

 Procedure.

1. HL-60 cells were harvested and washed in CRPMI. The counting of living cells was carried out using triptan blue. 3 • 10 ⁶ cells were placed in each of two 10 ml tubes, and 300 μl ⁵¹ Cr / 450 μCi / (New England Nuclear) was added to each tube. The tubes were incubated for 1 hour at a temperature of 37 ^o C with gentle stirring.

2. Antibodies were used as the upper layers of the hybridoma culture and they contained 0.01% NaN ₃ and 0.05% thimerosal. To remove these preservatives, 5 ml of each antibody was analyzed against 3 substitutions in 500 ml of each old tissue culture medium for 72 hours.

 3. Antibodies were collected after dialysis and 3.5 ml each was placed in 10 ml tubes. The residue was saved for use in the HL-60 binding ELISA. 7 ml of RPMI 1640 5% serum of the embryo of the cow was placed in a fourth tube for use as a control analysis.

 4. Labeled HL-60 cells were washed 3X in CRPMI and collected in one tube. Then they were subjected to centrifugation and again suspended in 1 ml of medium.

5. 200 μl of the cell suspension was added to each of the antibodies contained in the tube, and 400 μl in the control tube. The tubes were incubated for 20 minutes at 37 ^o C with gentle stirring.

 6. The plate with the analysis of stimulated HUYEK was removed from the incubator and the medium was removed from the well with a Pasteur pipette from several wells at the same time.

 7. 0.5 ml of cell suspension was added to each of the three recesses. Control cells were placed on plates on stimulated and unstimulated HUYEC at both concentrations of 1L-1β. Test cells were added only to stimulated wells.

 8. Portions of 0.5 ml of each cell suspension were added to glass tubes, which were then used to determine the input CHOM.

9. The assay plates were returned to the incubator / 5% CO ₂ , 37 ^° C. / for 30 minutes.

 10. A portion of each cell suspension was mixed with an equal volume of trypan blue and the cells were analyzed under a microscope for survivability. The following results were obtained: Control = 98%, SH1 = 92% and SNH3 = 99%.

 11. Unbound cells were removed from the recesses of the assay plate by systematic resuspension using a Pasteur pipette followed by addition and removal of 0.7 ml of medium.

12. The whole medium was removed from the recesses and a solution of 0.125 M Tris, 2% SDS (Bio-Rad / and 10% glycerol / Fisher / / 0.3 ml) was added. The plates were held for 30 minutes, and then 0.6 ml of dH ₂ O was added to each well.

 13. The fluid in each well was resuspended using a P1000 pipette and transferred to a glass tube. The P1000 tip was inserted into a test tube.

 14. Tubes, including those that contain input samples per minute / OM /, were analyzed on a gamma counter.

 15. The FOM associated with each well was divided by the input FOM for each sample to determine% binding. The mean and standard deviation points for the triple analysis were plotted on the coordinates.

 Replicates were considered the best in the experiment if high 1L-1 β was used in this experiment to induce endothelial cells.

 The results showed that the monoclonal antibody SNH3 blocked the binding of HL-60 cells to stimulated with 1L-1 β HUYEC via the ELAM-1 receptor. The SH1 control antibody, which does not bind the SLX determinant, did not block the binding of HL-60 cells to ELAM-1. This suggests that terminal sialic acid in the ligand is necessary for binding to ELAM-1.

B. Lane 2 monoclonal antibodies
Materials: Passage 3 HUYEC, which was grown on gelatin-coated assay plates with 48 wells (Costar), was used as a source of endothelial cells. The tablets were prepared as described previously. Two series of three recesses were left unstimulated as controls. Seven triples on each plate were stimulated with IL-1 β at a concentration of 30 μg / ml in 0.5 ml of EGM-UY. Cells were stimulated for exactly 4 hours. HL-60 cells / ATCC / were used as ligand-bearing cells. They were collected from bulk culture at CRPMI. The upper layers of fresh hybridoma containing monoclonal antibodies included: FH6 / IgM /, lower affinity mAb; SNH-3 / IgM / / 20 μg / ml /; SH-1 / IgG ₃ / / 10 μg / ml /; FH-2 / IgM /, mAb active relative to Le ^x ; SNH-4 / IgG3 /, high affinity antibody; and CSLEX-1 / IgM / / provided by Dr. P. Terasaki, UCLA as purified immunoglobulin at a concentration of 2.8 mg / ml diluted to 9 μg / ml in Modified Eagles Dalbecco Medium / DMEM / containing 5% SZK for use in this analysis. The specificities of these antibodies were as follows: FH6, SNH-4, SNH3, and CSLEX-1 were specific for SLX; FH2 and SH1 were specific for non-sialylated Le ^x .

 Procedure.

1. HL-60 cells were harvested and washed in CRPMI. The counting of living cells was carried out using triptan blue. 3 • 10 ⁶ cells were placed in each of two 10 ml tubes, and 300 μl ⁵¹ Cr / 450 μCi / (New England Nuclear) was added to each tube. The tubes were incubated for 1 hour at 37 ^o C with gentle stirring.

2. Labeled HL-60 cells were washed 3X in DMEM containing 5% CEC / hereinafter referred to as cDMEM / and collected in one tube. They were then centrifuged and resuspended in 4 x 10 ⁶ cells per ml in the same medium.

 3. 3.2 ml of the top layer of each monoclonal antibody culture and 3.2 ml of purified CSLEX-1/29 μg / were added to separate tubes; 6.4 ml of medium was added to the control tube.

4. 200 μl of a cell suspension (equivalent to about 8 • 10 ⁵ cells) was added to tubes containing these monoclonal antibodies, and 400 μl to a control tube. Then the tubes were incubated for 20 minutes at 37 ^o C with gentle stirring.

 5. The HUYEC stimulated assay plate was removed from the incubator and the wells were washed once with cDMEM and the medium was removed from the cavity using a Pasteur pipette from several wells at the same time.

 6. 0.4 ml of cell suspension was added to each well of one of the two plates. Control cells were applied to unstimulated and stimulated HUYEC. Antibody-treated cells were added only to stimulated wells.

 7. Portions of 0.4 ml of each cell suspension were added to glass tubes to be used to determine input PMR.

8. The assay plate was incubated at 37 ^° C. for 30 minutes.

 9. The remainder of each cell suspension and assay plates were placed in an ice bath for rapid cooling for 20 minutes.

10. Cell suspensions were layered onto a cooled plate, as was done above for plates at 37 ^° C. This plate was incubated for 30 minutes at 40 ^° C.

 The remaining stages of the analysis were carried out as described in stages 11-15 of section A above, with the exception that at stage 12 the plates were held for 15 minutes, not 30 minutes.

The results shown in FIG. 2A show that monoclonal antibodies SNH-3, FH6, SNH-4 and CSLEX-1, all of which are SLX-specific, significantly blocked the binding of HL-60 cells to ILU-1β-stimulated HUYEC via ELAM-1 receptor, when they were incubated at 37 ^o C. Monoclonal antibodies specific for Le ^x / FH2 and SH1 / were not effective inhibitors. Thus, the ligand for ELAM-1 contains a sialylated Le ^x antigen or a similar structure was found in cell surface glycoproteins or glycolipids.

After incubation at 4 ^o C / Fig. 2B / antibodies FH6 and SNH-3 / both IgM / enhanced binding. In these trials, significant agglutination of HL-60 cells in depressions was shown, which may be significant for this observation.

 C. Monoclonal antibodies block the adhesion of LEC11 cells to cells that express ELAM-1.

In this series of experiments, we analyzed the ability of monoclonal antibodies specific for SLX and for the non-sialylated form, Le ^x , to block adhesion with ELAM-1 LEC11 cells / that express SLX / and LEC12 cells / that express Le ^x / with IL-1 β HUYEC stimulated .

 Materials: Passage 4 HUYEC served as a source of endothelial cells. The plates were prepared exactly as described previously. Two series of triple depressions were left unstimulated as controls. 7 triples on each plate were stimulated with IL-1 β at a concentration of 30 μg / ml in a 0.5 ml volume of EGM-UV. Cells were stimulated exactly 4 hours. LEC11 and LEC12 cells, generally described by Stanley et al., J. Biol. Chem. T. 263, pp. 11374/1988 /, see above, were provided by Dr. P. Stanley. They were grown in suspension culture in complete alpha-MEM containing ribonucleotides and deoxyribonucleotides / Gibco /, penicillin / 100 units / ml / / streptomycin / 100 μg / ml / / Irvine Scientific /, L-glutamine / 2 mm / / company Irvine Scientific / and 10% SZK / Hazelton /. The monoclonal antibodies used in these experiments are described in Section B, above. These include: FH6, SNH-3, SH-1, FH-2, SNH-4, and CSLEX-1.

 Procedure.

1. LEC11 and LEC12 cells were harvested and washed in CRPMI. Living cells were counted using tryptane blue. 3 • 10 ⁶ cells of each cell line were placed in each of two 10 ml tubes, and 300 μl ⁵¹ Cr / 450 μCi / (New England Nuclear) was added to each tube. The tubes were incubated for 1 hour at a temperature of 37 ^o C with gentle stirring.

2. Radiolabeled cells were washed with X3 in cDMEM and collected in one tube. They were then centrifuged and resuspended in 4 x 10 ⁶ cells per ml in the same medium.

 3. 1.6 ml of the top layer of each monoclonal antibody and 1.6 ml of purified CSLEX-1/15 μg / were added to separate tubes; 3.2 ml of medium was placed in control tubes.

4. 200 μl of a cell suspension equivalent to 4 x 10 ⁵ LEC11 or LEC12 cells were added to tubes containing monoclonal antibodies and 400 μl to a control tube. The tubes were incubated for 20 minutes at a temperature of 37 ^o C with gentle stirring.

 5. The assay plate for stimulated HUYEC was removed from the incubator and the wells were washed once with cDMEM and the medium was removed from the wells with a Pasteur pipette from several wells at the same time.

 6. Cell suspensions and assay plate were placed in an ice bath to cool rapidly for 20 minutes.

 7. 0.4 ml of cell suspension was added to each well of the previously described assay plate. Control cells were plated on unstimulated and stimulated HUYEC. Antibody-treated cells were added only to stimulated wells. Each analysis was carried out in triplicate.

 8. Portions of 0.4 ml of each cell suspension were added to glass tubes to be used to determine input PMR.

9. The assay plate was incubated for 30 minutes at a temperature of 4 ^o C.

 The remaining stages of the analysis were carried out as described in stages 11-15 of Section A above, with the exception that at stage 12 the plates were held for 15 minutes.

The results shown in FIG. 3A and 3B indicate that monoclonal antibodies SNH-3, FH6, SNH-4 and SCLEX-1 / are all SLX-specific / substantially blocked the binding of LEC11 cells to IL-I β HUYEC-stimulated receptors via ELAM-1 receptor. LEC12 cells that do not express the SLX epitope did not bind activated endothelium. Monoclonal antibodies specific for Le ^x / NG2 and SH1 / caused weak inhibition of LEC11 binding.

Further confirmation that SLX is the main ligand for the ELAM-1 receptor was obtained by removing sialic acid from LEC11 and HL-60 cells. In these experiments, the treatment of LEC 11 and HL-60 cells before adhesion was analyzed using neuraminidase / sialidase / Clostridium perfringens, 1.6 u / ml / Type X, Sigma Chem., Co / for 90 minutes at 37 ^o C during ⁵¹ Cr-tagging. The results shown in Fig. 4 confirm that sialidase significantly reduced the adhesion of LEC 11 and HL-60 cells by 70-85% with activated endothelial cells.

 Example IV Glycosphingolipid liposomes block the binding of SLX cells to activated endothelial cells.

This Example describes the preparation of liposomes that contain various biosynthetically produced glycosphingolipids, on which the terminal carbohydrate is either SLX or Le ^x , or similar but not identical compounds. The ability of liposomes that contain SLX or SLX substitutes to block the binding of SLX expressing HL-60 cells and LEC11 cells to endothelial cells that have been previously stimulated to express ELAM-1 by treatment using IL-1 β has been shown.

 Materials

The glycosphingolipids used in this experiment are shown in table. 1; they were obtained from the Biomembran Institute, Seattle, BA, and were either purified or biosynthetically produced and characterized by NMR and mass spectrometry, as described generally in Hakomori, SI et al., J. Biol. Chem., T. 259, p. 4672/1984 / and Fukushi, J. et al., J / Biol. Chem. , t. 259, p. 10511/1984 /, which are hereby incorporated by reference. S-diLe ^x / SLX / were synthesized enzymatically by the addition of fucosyl residues using the Colo 205 cell line as the source of the enzyme and SH as the substrate. Unsialylated diLe ^{x was} similarly synthesized using nLc6 as substrate and cell line NC1 H-69. See Holmes et al., J. Biol Chem. t.260, p. 7619/1985 /, which is hereby incorporated by reference. SPG and SH were purified from bovine red blood cells with nLc6 produced by chemical removal of the terminal sialosyl residue from SH.

Liposomes containing glycosphingolipids were prepared as follows: 100 μg of glinolipid was added to 300 μg of phosphatidylcholine (Sigma, egg yolk) and 500 μg of cholesterol / Sigma / in chloroform-methanol / 2: 1 / and the whole solution was evaporated to dryness using N ₂ in 15 ml screw cap tubes.

 Passage 3 HUYEC, which was grown prior to fusion on a gelatin-coated assay plate with 48 recesses (Costar), was used as a source of endothelial cells. The plates were prepared as described previously. Two series of triple depressions were left unstimulated as controls. 14 triples of stimulation with IL-1β at a concentration of 30 μg / ml in a 0.5 ml volume of EGM-UV. Cells were stimulated exactly for 4 hours. HL-60 cells and LEC11 cells were cultured as described above.

 Procedure.

 1. One 48-well Costa costar recess containing HUYEC, grown prior to gelatin fusion, was removed from the incubator, and the medium in each well was removed using a Pasteur pipette and replaced with either 0.5 ml of fresh EGM-UY medium or the same medium containing 30 μg / ml IL-1β, and then the plate was returned to the incubator for 4 hours.

2. HL-60 and LEC11 cells were harvested and washed in CRPMI. Live cells were counted 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 two 10 ml tubes and 300 μl ⁵¹ Cr / 450 μCi / / New England Newclea / was added to each tube. The tubes were incubated for 1 hour at a temperature of 37 ^o C with gentle stirring.

3. Radiolabeled HL-60 and LEC11 cells were washed 3X in CRPMI and collected in one tube. Then they were centrifuged and again suspended to 2 • 10 ⁶ cells per ml in the same medium.

 4. A HUYEC-stimulated assay plate was removed from the incubator and the wells were washed twice with RPMI 1640 medium containing 5 mg / ml bovine serum albumin (ABS).

 5. Liposomes were prepared as follows. Evaporated tablets were dissolved in 100 μl of absolute ethanol and sonicated for 2 minutes. 2 ml of PBS was slowly added to the tubes over 2 minutes while continuing with sonication. This feed was diluted 1:10 in RPMI 1640 medium immediately before usev and 50 μl of a 100 mg / ml ABS feed solution was added to each 1 ml of diluted liposomes to provide a final concentration of 5 mg / ml ABS.

 6. The medium was removed from the recesses of the assay plate with a Pasteur pipette, from several recesses at the same time, and 0.3 ml of a suspension of liposomes was added to each of the 6 recesses stimulated with IL-1β. Liposome buffer containing ethanol, RPMI 1640 and ABS in the same concentrations as in the hollows containing liposomes was placed in control wells. The control buffer was applied to the plate on stimulated and unstimulated HUYEC. Samples containing liposomes were added to stimulated wells.

7. The plates were incubated for 40 minutes at 37 ^° C, and then 50 μl ⁵¹ Cr-labeled HL-60 or LEC11 cells were added to the wells. Each cell line was analyzed in those repeats for each liposome preparation. Final cell concentration was 10 ^6/350 .mu.l / well. Three portions of 50 μl of each cell suspension were added to glass tubes, which should be used to determine the input PMF, and the assay plate was incubated at 37 ^° C for 30 min.

8. Unbound cells were removed from the recesses of the assay plates by systematic re-suspension using a Pasteur pipette and then adding and removing 0.7 ml of medium. The whole medium was removed from the recesses and a solution of 0.125 M Tris, 2% SDS and 10% glycyrin / 0.3 ml / was added. The plates were held for 15 minutes, and then 0.5 ml of dH ₂ O was added to each well.

 9. The liquid in each well was again suspended and transferred to a glass tube. The pipette tip was inserted into the tube. Tubes, including those containing a sample with input FOM, were analyzed in a gamma counter. The FOM associated with each well was divided by the input FOM for each sample to determine% binding. The points of the mean and standard error of the analysis of triple repeats were plotted on the coordinates.

As shown in FIG. 5, liposomes containing selected glycolipids containing terminal sequences that contained SLX / S-diLe ^x , table 1 /, significantly inhibited the adhesion of HL-60 cells to activated endothelial cells at a temperature of 4 ^° C. Liposomes containing glycolipids with Le ^x / di-Le ^x / or other similar carbohydrate structures / Table 1 / showed minimal inhibition, which was independent of the structure of the carbohydrate group. Similar results were obtained with the adhesion of LEC 11 cells. When experiments were carried out at a temperature of 37 ^o C, the adhesion of HL-60 cells is reduced by liposomes containing glycolipids with SLX-structure / S-diLe ^x , 70% /, as well as to a lesser extent liposomes containing Le ^x / di-Le ^x , 40% /, which suggests that Le ^x may also interact with ELAM-1, but with a lower affinity. These experiments show that biosynthetically produced SLX or similar SLX substitute compounds, when included in liposome compositions, can serve as therapeutic compounds, for example, to reduce white blood cell infiltration at the site of inflammation.

 Dzhurkat cells bind to activated IL-1 endothelial cells mainly through a pair of adhesion Y-CAM / endothelial cell / - YLA-4 / cell Dzhurkat / / Wayner and others, J. Cell. Biol. t.109, p.1321 / in contrast to the fact that the adhesion of HL-60 and LEC 11 cells with activated endothelial cells is carried out through the ELAM-1 receptor. Dzhurkat cell adhesion was not inhibited using liposomes that contained SLX, but was completely inhibited by a monoclonal antibody with the α-subunit of the integrin molecule YLA-4. This result confirms that inhibition of HL-60 and LEC 11 cells by SLX liposomes is not a stearic effect attributed to the binding of liposomes to endothelial cells, but confirms the conclusion that SLX liposomes inhibit adhesion through direct competition with the ELAM-1 ligand binding point.

 Example V. Antibodies for SLX Inhibition of Binding with the Participation of GMP-140 on Activated Human Platelets.

In this example, the ability of monoclonal antibodies specific for SLX and non-sialylated Le ^x to block adhesion involving GMP-140 HL-60 cells with activated human platelets was studied.

Materials:
HL-60 cells are described above and have been used as a source of ligand-bearing cells. Dzhurkat cells were used as control, not carrying ligands. Used the same monoclonal antibodies SH-1, FH-2, SNH-4 and CSLEX-1, as described above.

Procedure:
1. Blood was taken from a normal human donor through a syringe containing an anticoagulant ACD / dextrose, 2.0 g; sodium citrate 2.49 g; and citric acid 1.25 g; up to 100 ml with dH ₂ O / in relation to 6 parts of blood per 1 part of anticoagulant.

Platelets were isolated by differential centrifugation as follows: Blood was centrifuged at 800 rpm / approximately 90 xg / for 15 minutes at room temperature. The top layer was collected and centrifuged at a speed of 1200 rpm / approximately 400 xg / 6 minutes. The top layer was removed and centrifuged at a speed of 2,000 rpm / 1200 xg / for 10 minutes to tablet platelets. This tablet 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; adjusted to 1 l with DI-water, pH 6.5 was obtained with an IN NaOH solution /, then washed once with SCK. Platelets were again suspended at a concentration of 10 ⁸ / ml in SZK.

2. About 20 minutes before the platelets were finally resuspended, 48-well plates were coated with 0.1% gelatin and incubated for 15 minutes at 37 ^° C. Excess gelatin was pipetted immediately before the platelet suspension was added. Platelets were activated by adding 0.25 units of thrombin / ml / Sigma, T-6759 / platelet suspension. Platelets were kept at room temperature for 20 minutes.

3. To obtain binding of activated platelets, 300 μl of platelet suspension was added to each well in a gel-coated plate. The plate was incubated at a temperature of 37 ^o C for 15 minutes, then processed at a speed of 800 rpm / 90 x g / for 2 minutes. Unbound platelets were removed by washing the plate 3 times using PBS.

4. Since platelets have highly active Fc receptors to prevent uptake of aggregated IgG from antibody preparation, platelet Fc receptors are blocked as follows. Purified murine IgG W6 / 32 / IgG _2a / at a concentration of 27 mg / ml was aggregated by heating to 63 ^° C for 5 minutes, 300 μl of a heated preparation at a concentration of 20 μg / ml in PBS was added to each well of a platelet-coated plate. The plate was incubated at 37 ^° C. for 15 minutes, then washed with PBS.

5. HL-60 and Jurcat cells were harvested and washed in CRPMI. Live cells were counted using trypan blue, 3 x 10 ⁶ cells of each type were placed in each of two 10 ml tubes, and 300 μl ⁵¹ Cr / 450 μCi // New England Newclea / were added to each tube. The tubes were incubated for 1 hour at a temperature of 37 ^o C with gentle stirring.

6. Radiolabeled cells were washed 3X in CRPMI and collected in one tube. Then they were centrifuged and again suspended to a concentration of 4 • 10 ⁶ cells per ml in the same medium.

 7. 1.6 ml of the top culture layer of each monoclonal antibody and 1.6 ml of purified CSLEX-1/15 μg / were added to separate tubes; control tubes received 1.6 ml of medium.

8. 100 μl of a suspension of labeled HL-60 cells or Jurcat cells / containing 4 • 10 ⁵ cells / was added to each of the tubes that contained the monoclonal antibody. They were incubated for 20 minutes at 37 ^o C with gentle stirring. After this incubation period, 0.3 ml of each cell suspension (containing 7.5 × 10 ⁴ cells) was added to each well of the previously described assay plate containing coupled activated platelets. Each analysis was carried out in triplicate.

9. The assay plate was centrifuged at 90 x g for 2 minutes, and then incubated for 5 minutes at room temperature. Unbound cells were removed from the recesses of the assay plate by inverting the plate in a radioactive dump receptacle and blotting the plate on towels. The wells were washed 3X by gently adding 300 μl of PBS to each well, and flipping and blotting the plate. The whole medium was removed from the recesses and 0.3 ml of a solution of 0.125 M Tris, 2% SDS and 10% glycerol was added. The plates were held for 15 minutes, and then 0.6 ml of dH ₂ O was added to each well.

 10. The fluid in each well was again pipetted and transferred to a glass tube. The tip was inserted into a test tube. Tubes, including those containing input samples, were analyzed in a gamma counter. FOMs associated with each well were divided into input FOMs for each sample to determine% binding. Input CHOM was determined by analyzing a portion of 0.3 ml of each cell suspension described in step 8.

The results shown in FIG. 6 show that monoclonal antibodies SNH-4 and CSLEX-1 specific for SLX blocked the binding of HL-60 cells to GMP-140 on activated platelets. Monoclonal antibodies specific for Le ^x / FH2 and SH-1 / also blocked this binding, but to a lesser extent. This Example suggests that both SLX and Le ^x may be ligands for GMP-140, but that the SLX structure may have a higher affinity for GMP-140 than the Le ^x structure.

 Example VI Glycosphingolipid liposomes block the binding of SLX cells to activated platelets.

 This example demonstrates the ability of liposomes that contain SLX or SLX analogues to block the binding of SLX-expressing HL-60 cells and PMN to platelets that were previously stimulated to express GMP-140 by treatment with thrombin. When performing analyzes in the General case followed the description given in Larsen et al. Cell, t. 63, p. 467-474 / 1990 /, which is incorporated herein by reference.

 Materials

 Glycosphingolipids were prepared as described in Example IV. Platelets were obtained as described in Example V with the exception that Fc receptors were not blocked. HL-60 cells were obtained as described above.

PMN was obtained from 50 ml of whole blood extracted from volunteer donors in Vacutainer heparin-treated tubes, which were inverted to mix the blood. All stages were carried out at a temperature of 22-24 ^o C. Every 25 ml of blood was applied in a layer of 15 ml of Mono-Poly dissolving Medium (Tlow Labs). The tubes were centrifuged at a speed of 800 x g for 25 minutes, then at a speed of 1300 x g for another 25 minutes. The PMN layer was removed and placed in a clean centrifuge tube of 50 cm ³ . Thirty ml of a balanced Hanks salt solution (Gibco company) containing 20 mM HEPE (Gibco company) and 0.2% glucose (Fisher company) was added to each tube, which was then centrifuged at a speed of 1900 x g for 3 min. PMN was washed 3X in the same buffer by centrifugation at a speed of 1900 x g for 3 minutes. ChOM was counted using a hemacytometer and resuspended to a concentration of 2 x 10 ⁶ / ml, then stored at room temperature until use.

 Procedure.

 1. 20 ul of activated platelet preparation was placed in each of 28 1.5 ml Eppendorf tubes / 14 double samples /.

 2. 20 ul diluted liposomes in 10 angles, 5 angles or 2 angles, or control buffers were added to the corresponding test tube of each two.

 3. Platelets were incubated with liposome preparations for 20 min at room temperature.

4. Neutrophils or HL-60 cells at a concentration of 2 • 10 ⁶ cells / ml each were added to one series of platelets treated with liposomes. 20 ul of cell suspension was added to each tube.

 5. The tubes were mixed and kept at room temperature for 20 minutes. Then they were applied to a hemacytometer and the cells were evaluated as positive / 2 and more platelets joined / cell / or negative / less than 2 platelets joined / cell /.

As shown in FIG. 7, liposomes containing selected glycolipids having terminal sequences that contained SLX / S-diLe ^x , Table 1 /, significantly inhibited the adhesion of HL-60 cells to activated platelets. Liposomes containing glycolipids with Le ^x / di-Le ^x / or other similar carbohydrate structures / Table 1 / showed minimal inhibition, which was independent of the structure of the carbohydrate group. Similar results were obtained with the adhesion of PMN cells (Fig. 8/). These experiments show that biosynthetically produced SLX or similar SLX substitutes, when included in liposome compositions, can serve as therapeutic compounds, for example, to reduce the binding of leukocytes to platelets at sites of inflammation.

 Example VII Oligosaccharides containing SLX block the binding of neutrophils to platelets.

In this example, the ability of the minimal SLX tetrasaccharide to inhibit GMP-140 adhesion was compared with the same property of penta- and hexasaccharides containing SLX. In short, platelets and neurophiles were isolated using the techniques previously described. Platelets were activated with thrombin and then incubated with dilutions of various oligosaccharides. Neutrophils were added and the effect of saccharides on the adhesion of neutrophils with activated platelets was determined. The following oligosaccharides were used: SLX (hexa), Neu Ac α 2,3 Gal β 1,4 (Fucα1,3) GlcNAc β 1,3 Gal β 1,4 Glc-O-CH ₂ CH ₂ -SiMe ₃ / invaluable gift from the professor Hasegawa, Gifu University, Japan /, SLX / Penta / Neu Ac α 2,3 Gal β 1,4 (Fuc α 1,3) Glc NAc β 1,3 Gal β and SLX / Tetra /, Neu Ac α 2,3 Gal β 1.4 (Fuc α 1.3) Glc NAc.

 Procedure.

1. Platelets were isolated as described above and activated / 2 • 10 ⁸ / ml / by incubation for 20 minutes at room temperature with thrombin at a final concentration of 0.25 u / ml.

 2. Neutrophils were isolated by applying a layer of heparin-treated blood to Mono-Poly solvent medium / Ficoll-Hypaque-Flow Laboratories, then centrifuged for 25 minutes at a speed of 2000 rpm, and then for another 25 minutes at a speed of 2500 rpm / min, as described above.

3. For analysis, 20 μl of platelet suspension / 2 • 10 ⁸ / ml / was placed in an Eppendorf centrifuge tube. An equal volume of oligosaccharide preparations was added in concentrations from 200 μg / ml to 0.3 μg / ml, or liposome-glycolipid preparations / obtained as described above / in concentrations from 2 μg / ml to 0.25 μg / ml, and tubes kept at room temperature for 20 minutes. Added 20 μl of neutrophil preparation / 2 • 10 ⁶ / ml / and the tubes were kept for another 20 minutes at room temperature.

 4. The adhesion of activated platelets to neutrophils was analyzed under a microscope. One hundred neutrophils were analyzed. They were rated as positive if 2 or more platelets joined, and negative if less than 2 platelets were bound. The percentage of cells with 2 or more associated platelets was calculated.

 The average values of the results of three identical experiments are given in table. 2.

As shown in the table. 2 above, about 20 times more SLX-tetrasaccharide is required for 50% inhibition of binding by GMP-140 neutrophils with thrombin-activated platelets than SLX-hexaccharide. The amount of tetrasaccharide required is approximately equal to the amount required for a similar degree of inhibition when non-sialylated Le ^{x is used} . The pentasaccharide gives 50% inhibition at concentrations similar to those required for 50% inhibition with hexasaccharide, which indicates that the minimum structure for maximum inhibition is closer to the pentasaccharide.

 Example VIII Adhesion blocking using a variant of SLX structures.

 This example describes experiments on testing various glycolipid structures. In particular, SV2, a sialylated polysaccharide in which fucose, instead of being attached to the last GlcNAc, as in SLX, was attached to the penultimate GlcNAc, was tested. Platelets and neutrophils were isolated using the techniques 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 glycolipids on the adhesion of neutrophils with activated platelets was determined.

The structures of the various glycolipids analyzed are shown below:
SDIY2, NeuGc α 2, 3Gal β 1,4 (Fuc α 1,3) GlcNac β 1,3Gal β 1,4 (Fuc α 1,3) GlcNAc β 1,3Gal β 1,4Glc β 1,1Cer; SLX, NeuGc α 2,3Gal β 1,4 (Fuc α 1,3) GlcNAc β 1,3Gal β 1,4Glc β 1,1Cer; SY2, NeuGc α 2,3Gal β 1,4GlcNAc β 1,3Gal β 1,4 (Fuc α 1,3) GlcNAc β 1,3Gal β 1,4Glc β 1,1Cer; SH, NeuGc α 2,3Gal β 1,4GlcNAc β 1,3Gal β 1,4GlcNAc β 1,3Gal β 1,4Glc β 1,1Cer; SPG, NeuGc α 2,3Gal β 1,4GlcNAc β 1,3Gal β 1,4Glc β 1,1Cer;
The results of two identical experiments are shown in table. 3.

 These results indicate that SY2 inhibited adhesion involving GMT-140 neutrophils with thrombin-activated platelets to the same extent as SLX and SDiY2 did.

 Example IX Block adhesion using other SLX options.

This example confirms that the affinity of sialylated Le ^x / SLX / relative to GMP-140 is the same regardless of whether the terminal sialic acid is in the form of N-acetyl neuraminate / NeuAc / or N-glycol neuraminate / NeuGc /. All materials were prepared as described above. Platelets and neutrophils were isolated in accordance with the described techniques. Platelets were activated with thrombin and then incubated with dilutions of various glycolipids contained in liposomes. Neutrophils were added and the effect of glycolipids on the adhesion of neutrophils with activated platelets was determined.

 The results of the experiment, in which synthetic SLX / Neu Ac / and SLX preparation obtained by enzymatic fucosylation of sialyl paragloboside purified from bovine red blood cells SLX / Neu Gc /, were directly compared, are shown in table. 4.

 These results show that SLX-hexasaccharides inhibited adhesion involving GMP-140 neutrophils with thrombin-activated platelets in the same manner, whether sialic acid Neu Ac or NeuGc. This result indicates that either the N-acetyl or N-glycoloyl derivative of sialic acid is recognized by GMP-140. Similar results were obtained using ELAM-1.

Different glycolipids were also tested in the same assay. The structures of the tested glycolipids are presented below:
SLX (hexa), NeuGc α 2,3Gal β 1,4 (Fuc α 1,3) GlcNAac β 1,3Gal β 1,4 Glс β 1,1 Ceramide; α 2,3 SLX cer, NeuAc α 2,3Gal β 1,4 (Fuc α 1,3) GlcNac β 1,3Gal β 1,4Glc β 1,1 Ceramide; α 2.6 SLX cer, NeuAc α 2.6Gal α 1.4 (Fuc β 1.3) GlcNAc α 1.3Gal β 1.4Glc β 1.1 Ceramide; SH, NeuGc β 2.3Gal α 1.4 GlcNAc β 1.3Gal β 1.4GlcNAc β 1.3Gal β 1.4Glc β 1.1 Ceramide.

 Example X Blocking adhesion using synthetic SLX.

 This example confirms that synthetic SLX binds ELAM-1 and inhibits neutrophil adhesion to activated endothelium. This example also shows that a chemical bond in sialic acid disrupts binding to ELAM-1.

 Received two synthetic compounds. One contained sialic acid in a β 2,3 bond, as in naturally occurring SLX. The second contained sialic acid in an α 2.6 linkage to explore the importance of the nature of the linkage with receptor binding.

Liposomes were prepared by adding 12 αl of absolute ETOH to each tube, rapidly heating in a water bath at 50 ^° C. and sonicating for 2 minutes. 238 μl of warm, phosphate-buffered saline (PBS) was slowly added to each tube, while sonication, which was then continued for another 10 minutes. The final concentration of raw liposomes was 400 μg glycolipids / ml in 5% ETOH / PBS.

 Procedure.

 1. HUYEC, PMN and liposomes were prepared as described above.

 2. A HUYEC-stimulated assay plate was removed from the incubator and the wells were washed twice with RPMI 1640 medium containing 5 mg / ml bovine serum albumin (ABS).

 3. The liposome feed was diluted in HBSS / ABS buffer to obtain solutions equivalent to: 40 μg / ml, 30 μg / ml, 15 μg / ml, 7.5 μg / ml, 3.75 μg / ml and 1.87 μg / ml Similar dilutions were obtained from control stock consisting of PBS-5% ETOH.

 4. The medium was removed from the recesses of the analytical tablet using a Pasteur pipette, from several recesses at the same time.

 5. 0.05 ml of a suspension of each liposome was added to double wells on stimulated assay plates. A liposome buffer containing ethanol HBSS and ABS at the same concentrations as in the wells containing liposomes was injected into the control wells. Control buffer was applied to the plate on unstimulated and stimulated HUYEC. Samples containing liposomes were added only to stimulated wells.

6. The plates were incubated for 40 minutes at 37 ^° C, and then 50 μl PMN was added to the assay wells. The final cell concentration was 5 • 10 ⁵ per well of 100 μl.

7. The assay plate was returned to the incubator / 5% CO ₂ , 37 ^° C / for 8 minutes.

 8. Unbound cells were removed from the recesses of the plates by systematic re-suspension using a p200 multichannel pipette, followed by addition and removal of 0.2 ml of medium.

 9. The whole medium was removed from the recesses and 50 μl of solubilization buffer was added. It consisted of citrate buffer / 24.3 ml 0.1 M citric acid, 10.5 g / 500 ml + 25.7 ml 0.2 M dibasic sodium phosphate, 14.2 g / 500 ml and then water to 100 ml / containing 0.1% detergent NO-40.

10. The plates were incubated on a rotary vibrator for 10 min, and then 0.05 ml of OPDA solution [8 mg of ortho-phenylene diamine, Sigma cat P-1528, 8 μl 30% H ₂ O ₂ and 10 was added to each well ml citrate buffer (as above)]. Reactions were allowed to develop for 15 minutes, and then 25 μl of a 4N sulfuric acid solution was added to each well to stop the reaction.

 11. The volume of reagents was obtained by displacing volumes of 100 μl of solubilization buffer and OPDA solution with 50 μl of 4N sulfuric acid solution.

 12. 100 μl of the upper layer was removed from each of the 2 recesses and transferred onto an ELISA elastic assay plate (Falcon). The tablet was scanned on a spectrophotometer at 492 nm for 30 minutes.

 The results of these two experiments are presented in table. 5.

These results show that liposomes containing synthetic α / 2, 3 / Cialyl Le ^x but not α / 2, 6 / Cialyl Le ^x inhibit neutrophil adhesion to activated endothelium in an ELAM-1 dependent binding assay. Thus, the α 2, 3 linkage of sialic acid is necessary for recognition by ELAM-1. In addition, these results show that the synthetically produced oligosaccharide, α / 2, 3 / Cialyl Le ^x , binds to ELAM-1 and blocks the binding of neutrophils to activated endothelium. Similar results were obtained for GMP-140 and they are presented in FIG. 9. Similar results were found with GMP-140. See FIG. 9. This compound or derivatives of this compound thus become potential candidates for an anti-inflammatory drug.

 Example XI Treatment of HL-60 cells with endo-β-galactosidase.

This example describes experiments to determine whether the internal sugar bond of the β-galactose backbone of sialylated Le ^x HL-60 cells is cleavable by Endo-β-galactosidase, an enzyme known to be used to cleave the internal β-galactose bond in polylactosaminyl structures, but not β -gal, when GlaNAc is attached to mannose / structures such as nuclei /.

 Procedure.

 Platelets were isolated and activated using thrombin using the techniques described above. Activated with 1l-1 β HUYEC was obtained as described above. The cultured HL-60 cells were treated with endo-β-galactosidase, as described below, and the effect of the enzyme treatment on adhesion with the participation of GMP-140 HL-60 activated platelet cells was determined.

The enzyme treatment of HL-60 cells was carried out as follows: 12.4 x 10 ⁶ cells were washed twice with a balanced solution of Hanks salts containing 20 mM HEPES and 0.2% glucose, then a single washing step was carried out in normal saline. Endo-β-galactosidase (0.1 unit, ICN Chemicals Inc., Irvine, Calif.) Was dissolved in 200 μl normal saline and 200 μl sodium acetate buffer, pH 6.01. 200 μl / containing 0.05 units of enzyme / was added to 3 • 10 ⁶ HL60 cells, and 200 μl of acetate buffer was added to the same number of cells, the latter was used as a buffer control. Both tubes were incubated at 37 ^° C. for 60 minutes with gentle shaking. Then the tubes were cooled in ice and the cells were washed three times in HBSS containing HEPES and glucose, then they were counted and suspended in 2 × 10 ⁶ / ml.

For analysis with GMP-140, 20 μl Tyrode-HEPES buffer, pH 7.2, was placed in an Eppendorf tube. The same volume of activated platelets / 2 • 10 ⁸ / ml / and HL-60/2 • 10 ⁶ / ml / cells were added and, after mixing, the tubes were kept at room temperature for 20 minutes. Platelet adhesion with HL60 cells was analyzed under a microscope, as described previously for the adhesion of activated platelets to neutrophils.

For ELAM-1 analysis, the enzyme treatment of HL-60 cells was carried out as described above, with the exception that these cells were simultaneously labeled with ⁵¹ Cr, as described previously. Adhesion involving ELAM-1 was stopped by incubating 2 × 10 ⁵ treated or untreated cells with activated IL-1HUYEC for 30 minutes at 4 ^° C, and then washing the plate with a Pasteur pipette.

The results of these experiments showed that treatment of HL-60 cells with endo-β-galactosidase inhibited their ability to bind to / 1 / thrombin-activated platelets by 87.5% and / 2 / activated by 1L-1 β HUYEC at a temperature of 4 ^o C by 70% . Thus, the minimal SLX-containing tetrasaccharide ligand for GMP-140 probably attaches to the lactose or polylactosaminyl structure rather than to mannose.

 Example XII. Fucosylated polysaccharide blocks the binding of neutrophils to platelets.

In this example, the ability of a fucosylated polysaccharide to inhibit adhesion with GMP-140 was compared with the same properties of unfucosylated polysaccharide, SLX hexasaccharide, and Le ^x . In short, platelets and neutrophils were isolated using the procedures described above. Platelets were activated with thrombin and then incubated with dilutions of various oligosaccharides. Neutrophils were added and the effect of saccharides on the adhesion of neutrophils with activated platelets was determined. Used oligosaccharides: Native polysaccharide and its fucosylated derivative / preparation of both described below /; SLX-hexasaccharide, LNF III / Le ^x / and LNF1 / structure described above /.

 The conversion of the polysaccharide, which contains the structure of the linear core of SLX, into a multivalent, containing SLX, polysaccharide was carried out using enzymatic fucosylation. A native type 1a polysaccharide was obtained from Group B Streptococcus as described in Iennings et al., Biochem. T. 22, p. 1258-1263 / 1983 /, which is hereby incorporated by reference. The corresponding bacterial strains are deposited with the American Type Culture Collection / ATCC / under storage numbers NN 12400, 31574, 12401 and 31575.

To obtain a fucosylated polysaccharide, a native polysaccharide of type 1a / 1 mg / was dissolved in a mixture of 6 μl of 1 M manganese chloride, guanosine 5'-diphosphate β-L-fucose with a radiolabeled indicator / specific activity 1.82 • 10 ⁶ counts μmol /, 0 , 9 μmol in water 90 μl and water 137 μl. To this mixture was added 100 μl of a 3/4 solution of fucosyl transferase isolated from human milk, as previously described by Prieels et al., J. Biol. Chem. T. 256, pp. 10456-10463 / 1981 /, which is hereby incorporated by reference.

 The reaction mixture was concentrated against a membrane / cutting off 100 K / several times with water, and what was left was lyophilized to obtain a powder. This solid was dissolved in water and passed through a weak cation exchange column to remove any remaining protein. Radioactive fractions containing fucosylated polysaccharide were collected and lyophilized. About fifty of the available side chains were fucosylated, which was established by the introduction of RFID tags.

 Procedure.

Platelets were isolated as described above and activated / 2 • 10 ⁸ / ml / by incubation for 20 minutes at room temperature with thrombin at a final concentration of 0.25 U / ml.

 Neutrophils were isolated by forming a layer of heparin-treated blood on a Mono-Poly dissolving medium (Ficoll-Hypague, Flow Zaboratories), and then centrifuged for 25 min at a speed of 2000 rpm, and then another 25 minutes at a speed of 2500 rpm as described above.

For this analysis, 20 μl of platelet suspension / 2 • 10 ⁸ / ml / was placed in an Eppendorf centrifuge tube. An equal volume of oligosaccharide preparations was added in concentrations from 500 μg / ml to 2.0 μg / ml, and the tubes were kept at room temperature for 20 minutes. Then added twenty μl of neutrophil preparation / 2 • 10 ⁶ / ml / and the tubes were kept for another 20 minutes at room temperature.

 The adhesion of activated platelets to neutrophils was analyzed under a microscope. One hundred neutrophils were studied. They were evaluated as positive if 2 or more platelets were attached, and as negative if less than 2 platelets were bound. The percentage of cells with 2 or more platelets bound was calculated.

 As shown in table 6, fucosylated polysaccharides very effectively inhibited the binding of neutrophils to thrombin-activated platelets involving GMP-140-50% inhibition was achieved with less than 1 μg / ml. This was compared with 20 μg / ml - a native polysaccharide was needed at that concentration, and with 8 μg / ml - SLX-hexasaccharide was needed at that concentration for a similar degree of inhibition.

 Example XIII Protection of rats from endotoxic shock / death / using monoclonal antibodies against LAM-2.

 This example confirms the efficacy of the P6E2 mAb / mouse 1gG-3k, a functional anti-human ELAM-1 mAb described in U.S.S.N. 07/645878, which is simultaneously pending, which is hereby incorporated by reference / in an animal model of lipopolysaccharide-induced death. The rat system was chosen because P6E2 was shown to cross-interact with the rat equivalent of ELAM-1.

 Materials and methods.

 LPS from E. coli 0111: B4 / Sigma / was obtained fresh from one batch one day before use by dissolving a sterile, pyrogen-free, saline solution at a concentration of 5 mg / ml. The solution was sonicated on ice for 30 seconds using a Tekmark / Tekmark / sonic cell disruptor. Immediately before use, the materials were sonicated a second time for 30 seconds.

 Female Lewis rats weighing 200 g (± 10 g / received from Charles River Breeding Labs and kept for at least 7 days after receiving / for adaptation /. Used groups / 10 animals /, if not explicitly indicated otherwise. All reagents they were injected parenterally through the tail vein, and animals that were given either sterile, LPS-free saline or mouse IgG 3k myeloma protein 3K / J 606, low progen <2 ng / mg protein / were used as negative control animals.

 Results.

The P6E2 dose / schedule protocols were obtained empirically from the pharmacokinetic data that were obtained when used with P6E2 prophylactic targets in rats. The minimum LD ₁₀₀ dose was determined empirically and it was 7.5 mg / kg for these rats.

 In one experiment, rats were treated with a dose of 10 mg / kg one hour prior to LPS administration. The drug was administered 3 hours after LPS. 4/10 treated animals survived. On the contrary, all 10 control animals / which were injected with saline / died. During the observation period of twenty-four hours, some clinical signs characteristic of the treated LPS animals could be observed in the surviving animals.

 The applicant further tested doses of P6E2 that were / 1 / twice the dose from the first experiment, and / 2 / the order of magnitude was lower than 10 mg / kg, which was used in the first experiment. The results show that P6E2 had a significant effect: 80% of the animals survived at a dose of 10 mg-kg / Fig. ten/. Note that one animal survived in the control group of saline - which suggested that this group of animals was not "the most successful" in terms of the severity of the outcome.

 Another study was carried out in order to confirm the therapeutic value of P6E2. Animals were given a feed dose of 10 mg / kg P6E2 1 hour before or 2, 4 or 6 hours after administration of LPS.

 Again, 1 out of 10 animals survived in the group treated with saline, as well as in the group treated with a dose of 10 mg / kg of myeloma protein J 606 at T = -60 minutes / Fig. eleven/. P6E2 had a protective effect, even when it was applied 2 or 4 hours after LPS.

 Conclusion

 The protection noted for Cytel mAb P6E2 confirms the importance of ELAM-1 in animal models of fatal diseases.

 Example XIV. "Rolling" SLEX liposomes on activated 1L-1 endothelial rabbit cells.

 Goal.

 This experiment confirms that sialylated Lewis / SLX / and sialoparagloboside / SPG /, isolated from biological sources and included in liposomes, will "roll" along activated rabbit venules.

 "Rolling" is an early intercellular interaction between white blood cells and the walls of endothelial cells. The white blood cell will "roll" on the endothelial cells. Further, the leukocyte will either / 1 / be released again into the circulation, or / 2 / “stick” to the endothelial cell, and early events that end with inflammation begin. Selectins were the result of cell-to-cell interactions during rolling, see von Adrian et al., Proc. Natl. Acad Sci. USA, 1991.

 Materials

Liposomes containing glycosphingolipids were prepared as follows: 50 g of glycolipid was added to 150 g of phosphatidylcholine (Sigma, egg yolk /, 250 g of cholesterol / Sigma / and 1 mM carboxyfluorocein / Sigma / in a mixture of chloroform: methanol / 2: 1 / and the whole solution was evaporated to dryness in glass tubes with screw caps. Liposomes were prepared by adding 12.5 Ul of absolute ethanol to each tube, brief heating and water bath and sonication for 2 minutes. 238 ul of warm PBS was slowly added to each tube while being sonicated, and this treatment was continued for only 10 minutes. Liposomes were transferred in 1 ml with PBS and centrifuged at 14,000 rpm for 2 minutes in order to remove excess EtOH and carboxyfluoroacein. The top layer was discarded, and the liposomes were again suspended in 1 ml of PBS, analyzed on a hemacytometer and adjusted to provide 5 x 10 ⁶ liposomes per ml. One ml was injected into a rabbit “activated” 4 hours earlier with 1L-1.

“Rolling” was observed using a vital microscope, as described by von Adrian et al. The mesentery of a 1L-1 activated rabbit was temporarily removed and placed on a glass window heated to 37 ^° C on a microscope stage. The mesentery was lowered into the bath at a temperature of 36.5 ^° C with brine balanced with 5% CO ₂ in N ₂ . Liposome-endothelial interactions were observed using a vital microscope with a 50X objective when immersed in salt water. Liposomes were made observable by fluorescent light. In one minute, liposomes were counted in the segment of the selected mesenteric venule / 20-40 μm in diameter /.

Results. Liposome Rolling in Rabbit Cells Activated with 1L-1
Injectable material - rolling
human neutrophils - 4 / maximum number of rolls /
SLex liposomes - 2 \\\ SPG liposomes - 0 / no rolling /
Conclusion

 In addition to LAM-1 / von Andrian et al. /, ELAM-1 and its ligands, as it turned out, are included in the process of leukocyte accumulation along the edges or “rolling”.

 Although the above invention has been described in some detail by way of illustration and example for purposes of clarity, it should also be clear that some changes and modifications may be made while remaining within the scope of the appended claims.

Claims (77)

1. A pharmaceutical composition based on an intercellular adhesion mediator comprising a pharmaceutically acceptable carrier and a compound containing a component that selectively binds to a selectin receptor and which has the formula
R ¹ -Galβ1,4 (Fucα1,3) GlcNAc- (R ² ) _a -X,
in which R ¹ is an element selected from the group consisting of sialic acid, an oligosaccharide containing sialic acid and a group having the formula

in which R ³ and R ⁴ , taken separately, which may be the same or different, are selected from the group consisting of H, C ₁ -C ₈ alkyl, hydroxy (C ₁ -C ₈ alkyl), aryl (C ₁ -C ₈ alkyl) and (C ₁ -C ₈ alkoxy) - (C ₁ -C ₈ alkyl); or
R ³ and R ⁴ taken together form a 4 to 8 membered ring;
R ² is β1, 3Gal, α1,2Man or α1,6GalNAc and a is 0 or 1,
X is H, lower (C ₁ -C ₈ ) alkyl, arylalkyl, hydroxy (C ₁ -C ₈ ) alkyl or (C ₁ -C ₈ ) alkoxy- (C ₁ -C ₈ ) alkyl.  2. The composition according to claim 1, characterized in that the compound is a biomolecule.  3. The composition according to claim 2, characterized in that the biomolecule is an oligosaccharide, oligopeptide, protein or lipid. 4. The composition according to claim 1, characterized in that R ³ and R ^{4 are} connected to form a 4 to 8 membered ring.  5. The composition according to claim 4, characterized in that the 4 to 8 membered ring is a monosaccharide.  6. The composition according to claim 1, characterized in that the sialic acid is NeuAcα2.3 or NeuGcα2.3.  7. The composition according to p. 1, characterized in that the oligosaccharide is NeuAcα2,3 Galβ1,4 Glc NAcβ1,3 or NeuGcα2,3 Galβ1,4 Glc NAcβ1,3.  8. The composition according to claim 1, characterized in that the compound is a polysaccharide. 9. The composition of claim 8, wherein the polysaccharide contains a repeating unit having the formula

10. The composition of claim 8, wherein the polysaccharide contains a repeating unit having the formula

11. The composition of claim 8, wherein the polysaccharide is a fucosylated polysaccharide of Group B Streptococcus type Ia.  12. The composition according to claim 8, characterized in that the polysaccharide is a polysaccharide of Streptococcus Group B, type II.  13. The composition of claim 8, wherein the polysaccharide has a molecular weight in the range of from about 5000 to 300,000 daltons.  14. The composition according to p. 8, characterized in that the polysaccharide contains from about 5 to about 200 fucosylated repeating units.  15. The composition according to p. 14, characterized in that the polysaccharide contains from about 25 to about 100 fucosylated repeating units.  16. The composition according to p. 2, characterized in that the biomolecule is a sphingolipid.  17. The composition according to p. 16, characterized in that the biomolecule is a ganglioside.  18. The composition according to claim 1, characterized in that the selectin receptor is expressed on vascular endothelial cells or platelets.  19. The composition according to p. 18, characterized in that the selectin receptor is ELAM-1 or GMP-140.  20. A pharmaceutical composition, characterized in that it contains a pharmaceutically acceptable carrier and a liposome that contains a compound that selectively binds selectin receptor in an effective amount.  21. The composition according to claim 20, characterized in that the liposome contains an anti-inflammatory chemotherapeutic agent.  22. The composition according to item 21, wherein the anti-inflammatory agent is cyclosporin A, indomethacin, naproxen, FK-506 or mycophenic acid. 23. The composition according to claim 20, characterized in that the compound has the formula
R ₁ -Galβ1,4 (Fucα1,3) GlcNAc- (R ₂ ) _a -Y
in which R ₁ selected from the group consisting of

NeuGcα2.3Galβ1.4GlcNAcβ1.3,
R ₂ is β 1,3Gal, α 1,2 Man or α 1,6GalNAc, and a is 0 or 1;
Y is a protein or lipid.  24. The composition according to item 23, wherein Y is a glycoprotein having a molecular weight of from 40,000 to about 250,000 daltons.  25. The composition according to item 23, wherein Y is a glycolipid having a molecular weight of from about 600 to about 4000 daltons. 26. The composition according to claim 20, characterized in that the compound contains a component having the formula
NeuAcα2.3Galβ1.4 (Fucα1.3) GlcNAc- (R) _a -X,
NeuGcα2,3Galβ1,4 (Fucα1,3) GlcNAc- (R) _a -X,
NeuGcα2,3Galβ1,4GlcNAcβ1,3Galβ1,4 (Fucα1,3) GlcNAc-R) _a -X,
in which R is β 1,3 Gal, α 1,2 Man or α 1,6 Gal A and a is 0 or 1,
X is H, lower alkyl (C ₁ -C ₈ ), arylalkyl, hydroxy- (C ₁ -C ₈ ) alkyl or (C ₁ -C ₈ ) alkoxy- (C ₁ -C ₈ ) alkyl.  27. The composition according to claim 20, characterized in that the selectin receptor is expressed on vascular endothelial cells or platelets. 28. A pharmaceutical composition, characterized in that it contains a pharmaceutically acceptable carrier and a compound that selectively binds a selectin receptor, the compound having the formula
R ₁ -Galβ1,4 (Fucα1,3) GlcNAc- (R ₂ ) _a -X,
in which R ₁ is sialic acid, R ₃ (R ₄ ) C (CO ₂ H) -, or a trisaccharide containing sialic acid;
R ₃ and R ₄ , which may be the same or different, are H, C ₁ -C ₈ alkyl, hydroxy-substituted C ₁ -C ₈ alkyl, aryl-substituted C ₁ -C ₈ alkyl or alkoxy substituted C ₁ -C ₈ alkyl;
R ₂ is β 1,3Gal, α 1,2 Man or α 1,6GalNAc and a is 0 or 1;
X is selected from the group consisting of H, lower (C ₁ -C ₈ ) alkyl, hydroxy (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) alkoxy (C ₁ -C ₈ ) alkyl, aryl, alkylaryl and arylalkyl. 29. The composition according to p. 28, characterized in that the compound has the formula
NeuAcα2.3Galβ1.4 (Fucα1.3) GlcNAc- (R) _a ,
NeuGcα2,3Galβ1,4 (Fucα1,3) GlcNAc- (R) _a ,
NeuGcα2,3Galβ1,4GlcNAcβ1,3Galβ1,4 (Fucα1,3) GlcNAc-R) _a ,
in which R is β1,3 Gal, α1,2 Man or α1,6 GalNAc and a is 0 or 1. 30. A pharmaceutical composition, characterized in that it contains a pharmaceutically acceptable carrier and a compound having two or more repeating units capable of selectively binding to a selectin receptor, wherein the repeating units contain a selectin binding moiety and are linked via a linker moiety, each repeating unit having the formula
R ₁ -Galβ1,4 (Fucα1,3) GlcNAc- (R ² ) _a -X,
in which R ¹ is a part selected from the group consisting of sialic acid, an oligosaccharide containing sialic acid, and a group having the formula

in which R ³ and R ⁴ taken separately, which may be the same or different, are selected from the group consisting of H, C ₁ -C ₈ alkyl, hydroxy- (C ₁ -C ₈ alkyl), aryl (C ₁ - C ₈ alkyl) and (C ₁ -C ₈ alkoxy) - (C ₁ -C ₈ ) alkyl; or
R ³ and R ⁴ taken together form a 4 to 8 membered ring,
R ² is β1,3 Gal, α1,2 Man or α1,6 GalNAc, and a is 0 or 1;
X is H, lower (C ₁ -C ₈ ) alkyl, arylalkyl, hydroxy (C ₁ -C ₈ ) alkyl or (C ₁ -C ₈ ) alkoxy (C ₁ -C ₈ ) alkyl. 31. The composition according to p. 30, characterized in that the linker component has the formula

in which n and m, which may be the same or different, are integers from 2 to 12; Y is O or S; and W is O, S or NH. 32. The composition according to p. 30, characterized in that the linker component is a 5 to 14-membered ring having two substituents, each Deputy has the formula

in which Y is O or S; a substituents are in the cis or trans position.  33. The composition according to p, characterized in that the substituents are in 1,2 - 1, [p / 2] + 1 position, where p is an integer from 5 to 14 and corresponds to the size of the ring. 34. A pharmaceutical composition, characterized in that it contains a pharmaceutically acceptable carrier and a heterocyclic compound containing two nitrogen atoms and two selectin binding components, each component being attached to one of the nitrogen atoms and has the formula
R ₁ -Galβ1,4 (Fucα1,3) GlcNAc- (R ₂ ) _a ,
in which R ₁ is sialic acid, R ₃ (R ₄ ) C (CO ₂ H) -, or a trisaccharide containing sialic acid,
where R ₃ and R ₄ , which may be the same or different, are H, C ₁ -C ₈ alkyl, hydroxyl substituted C ₁ -C ₈ alkyl, aryl substituted C ₁ -C ₈ alkyl, or alkoxy substituted C ₁ -C ₈ alkyl;
R ₂ is β 1,3Gal, α1,2 Man or α1,6GalNAc and a is 0 or 1.  35. The composition according to clause 34, wherein the heterocyclic compound is piperazine or homopiperazine. 36. A pharmaceutical composition, characterized in that it contains a pharmaceutically acceptable carrier and an amino acid associated with an oligosaccharide component that binds selectin selected from the group consisting of
NeuAcα2.3Galβ1.4 (Fucα1.3) GlcNAc- (R) _a -,
NeuGcα2,3Galβ1,4 (Fucα1,3) GlcNAc- (R) _a -,

in which R is β 1,3Gal, α 1,2 Man or α 1,6 GalNAc and a is 0 or 1.  37. The composition according to p. 36, wherein the amino acid is lysine, homolysin, ornithine, diaminobutyric acid, asparagine or diaminopropionic acid.  38. The composition according to clause 36, wherein the amino acid is included in the oligopeptide.  39. The composition according to p. 38, wherein the oligopeptide contains one or more compounds from the following group: lysine, homolysin, ornithine, diaminobutyric acid, asparagine or diaminopropionic acid.  40. The composition according to § 39, wherein the oligopeptide further comprises one or more compounds from the following group: alanine, tyrosine or radioiodinated tyrosine. 41. The composition according to p. 38, characterized in that the oligopeptide contains in the direction from the N-terminus to the C-terminus

in which R ₁ and R ₂ , which may be the same or different, are any amino acid residue, and L is the oligosaccharide part.  42. A pharmaceutical composition for treating an inflammatory disease, characterized in that it comprises a pharmaceutically acceptable carrier and an immunoglobulin capable of selectively binding to an oligosaccharide ligand recognizable by a cell surface selectin receptor, the immunoglobulin being contained in an amount effective to treat the disease. 43. The composition according to item 42, wherein the ligand is selected from the group consisting of
NeuAcα2.3Galβ1.4 (Fucα1.3) GlcNAc- (R) _a -,
NeuGcα2,3Galβ1,4 (Fucα1,3) GlcNAc- (R) _a -,

in which R is β 1,3 Gal, α 1,2 Man or α 1,6 GalNAc and a is 0 or 1.  44. The composition of claim 42, wherein the oligosaccharide moiety is expressed by a white blood cell.  45. The composition according to § 42, wherein the selectin receptor is expressed by a vascular endothelial cell or platelet.  46. The composition according to § 42, wherein the selectin receptor is ELAM-1 or GMP-140.  47. The composition according to item 42, wherein the composition is in the form of a single dose. 48. A pharmaceutical composition, characterized in that it contains a pharmaceutically acceptable carrier and a compound containing a component that selectively binds to a selectin receptor, this component having the formula
R ₁ -Galβ1,4 (R ₂ ) GlcNac- (R ₃ ) _a -X,
in which R ₁ is NeuAc α2,3, NeuGc α2,3, NeuAcα 2,3, Galα 1,4 GlcNAc β 1,3, or NeuGc β2,3 Gal α1,4 Glc A β1,3;
R ₂ is Fuc β 1,3, Aragal 1,3, / R, S / -5-alkyl-Ara α 1,3 and / R, S / -5-aryl-Ara α1,3;
R ₃ is 1.3 β Gal, 1.2 αMan or 1.6 αGalNAc and a is 0 or 1;
X is H, lower (C ₁ -C ₈ ) alkyl, arylalkyl, hydroxy (C ₁ -C ₈ ) alkyl or (C ₁ -C ₈ ) alkoxy (C ₁ -C ₈ ) alkyl.  49. The composition according to p, characterized in that the compound is a biomolecule.  50. The composition according to p, characterized in that the component binds to a selectin receptor expressed on a vascular endothelial cell or platelet.  51. The composition according to p, characterized in that the selectin receptor is ELAM-1 or GMP-140.  52. A method of inhibiting intercellular adhesion in a patient mediated by selectin, characterized in that it comprises administering a therapeutically effective dose of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound that selectively binds to a selectin receptor. 53. The method according to paragraph 52, wherein the compound contains a component that selectively binds to a selectin receptor, and this component has the formula
R ₁ -Galβ1,4 (Fucα1,3) GlcNAc- (R ₂ ) _a -X,
in which R ₁ is sialic acid, R ₃ (R ₄ ) C (CO ₂ H) - or a trisaccharide containing sialic acid;
where R ₃ and R ₄ , which may be the same or different, are H, C ₁ -C ₈ alkyl, hydroxy-substituted C ₁ -C ₈ alkyl, aryl-substituted C ₁ -C ₈ alkyl or alkoxy substituted C ₁ -C ₈ alkyl; and
R ₂ is β 1,3Gal, α 1,2 Man or α 1,6GalNAc and a is 0 or 1;
X is H, lower (C ₁ -C ₈ ) alkyl, arylalkyl, hydroxy (C ₁ -C ₈ ) alkyl or (C ₁ -C ₈ ) alkoxy (C ₁ -C ₈ ) alkyl. 54. The method according to paragraph 52, wherein the compound is (C ₁ -C ₈ ) alkyl, (C ₁ -C ₈ ) alkoxy (C ₁ -C ₈ ) alkyl.  55. The method according to paragraph 52, wherein the intercellular adhesion is associated with an inflammatory disease.  56. The method according to clause 55, wherein the inflammatory disease is septic shock.  57. The method according to clause 55, wherein the inflammatory disease is an acute respiratory syndrome syndrome or sepsis associated with a wound.  58. The method according to paragraph 52, wherein the intercellular adhesion is associated with metastases.  59. The method according to paragraph 52, wherein the selectin receptor mediates the adhesion of a white blood cell, monocyte or neutrophil to an endothelial cell.  60. The method according to paragraph 52, wherein the selectin receptor is ELAM-1 or GMP-140.  61. The method according to paragraph 52, wherein the compound is enclosed in a liposome.  62. The method according to paragraph 52, wherein the compound is a polysaccharide. 63. A method for treating an inflammatory disease mediated by a selectin receptor in a patient, characterized in that it comprises administering a therapeutically effective dose of a compound that selectively binds to this receptor and has the formula
R ₁ -Galβ1,4 (Fucα1,3) GlcNAc- (R ₂ ) _a -X,
in which R ₁ is sialic acid, R ₃ (R ₄ ) C (CO ₂ H) or a trisaccharide containing sialic acid;
where R ₃ and R ₄ , which may be the same or different, are H, C ₁ -C ₈ alkyl, hydroxy-substituted C ₁ -C ₈ alkyl, aryl-substituted C ₁ -C ₈ alkyl or alkoxy substituted C ₁ -C ₈ alkyl; and
R ₂ is ββ 1,3Gal, α 1,2 Man or α 1,6 GalNAc and a is 0 or 1;
X is H, lower (C ₁ -C ₈ ) alkyl, arylalkyl, hydroxy (C ₁ -C ₈ ) alkyl or (C ₁ -C ₈ ) alkoxy- (C ₁ -C ₈ ) alkyl. 64. The method according to p, characterized in that R ₁ is sialic acid, R ₂ is β 1,3 Gal, a is l, and X is (C ₁ -C ₈ ) alkoxy (C ₁ -C ₈ ) alkyl .  65. The method according to p, characterized in that the selectin receptor is ELAM-1 or GMP-140.  66. A method of analyzing a test compound for its ability to inhibit cell adhesion with selectin, characterized in that it comprises contacting the test compound with a selectin receptor and an isolated selectin binding agent and detecting the ability of the test compound to inhibit receptor and agent binding.  67. The method according to p, characterized in that the agent contains an SLX component or SLX substitute.  68. The method according to p, characterized in that the receptor or agent is immobilized on a solid surface.  69. The method according to p, characterized in that the test compound is an oligosaccharide or glycoconjugate.  70. A pharmaceutical composition, characterized in that it contains a pharmaceutically acceptable carrier and a compound capable of blocking cell adhesion with selectin, the compound being identified by contacting the test compound with a selectin receptor and an isolated selectin receptor binding agent, and the identified compound is capable of inhibiting binding between the receptor and agent. 71. A method of obtaining a compound containing an oligosaccharide component capable of selectively binding to a selectin receptor, characterized in that it involves fucosylation of a polysaccharide containing a sequence having the formula
R ₁ -Galβ1,4GlcNAc- ,,
in which R is sialic acid.  72. The method according to p, characterized in that the stage of fucosylation is carried out using fucosyl transferase.  73. The method according to p, characterized in that the polysaccharide is a polysaccharide of Streptococcus Group B type Ia.  74. The method according to p, characterized in that the polysaccharide is a polysaccharide of Streptococcus group B type II.  75. A method of obtaining a compound containing many components capable of selectively binding to a selectin receptor, characterized in that it involves the binding of these components together using a linker component. 76. The method according to item 75, wherein the components are selected from the group consisting of
R ₁ -Galβ1,4 (Fucα1,3) GlcNAc- (R ₂ ) _a ,
in which R ₁ is sialic acid, R ₃ (R ₄ ) C (CO ₂ H) - or a trisaccharide containing sialic acid;
where R ₃ and R ₄ , which may be the same or different, are H, C ₁ -C ₈ alkyl, hydroxy-substituted C ₁ -C ₈ alkyl, aryl-substituted C ₁ -C ₈ alkyl or alkoxy substituted C ₁ -C ₈ alkyl;
R ₂ is β1,3Gal, α1,2 Man or α1,6 Gal NAc and a is 0 or 1. 77. The method according to item 75, wherein the linker component has the formula

in which n and m, which may be the same or different, are integers from 2 to 12; Y is 0 or S; and W is O, S or NH. 78. The method according to item 75, wherein the linker component is a 5 to 14-membered ring having two substituents, each Deputy has the formula

in which Y is O or S; and the substituents are in the cis or trans position.  79. The method according to item 75, wherein the substituents are in 1,2 - 1, [p / 2] + 1 position, where p is an integer from 5 to 14, and corresponds to the size of the ring.

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