IE912650A1 - Inhibitors of fucosyltransferase and their uses - Google Patents

Abstract

The present invention relates to inhibitors of 5 fucosyltransferases involved in the synthesis of carbohydrate ligands recognized by selectin cell surface receptors. Screening methods to identify these compounds and pharmaceutical uses are provided.

Description

INHIBITORS OF FUCOSYLTRANSFERASE AND THEIR USES 5 Field of the invention The present invention relates to compositions and methods for treating inflammation and inflammatory disease processes and other pathological conditions mediated by intercellular adhesion. In particular, it relates to inhibition of cellular adhesion using blocking agents that selectively inhibit fucosyltransferase, an enzyme involved in the synthesis of cell surface glycoproteins and glycolipids associated with intercellular adhesion.

BACKGROUND OF THE INVENTION Intercellular recognition and adhesion is a complex phenomenon necessary for numerous cellular interactions, such as fertilization, cell migration, organ formation, and immune defense. The high selectivity required by these processes is often provided by lectins, a class of nonimmunogenic proteins that bind carbohydrates selectively and noncovalently.

Typically, lectins recognize and bind carbohydrates associated with proteins and lipids on the cell surface of the apposing cell.

Such intercellular protein-carbohydrate interactions are thought to be associated with the interaction of vascular endothelial cells and various circulating cells in the blood stream. The vascular endothelium plays a key role in binding certain cells in the blood stream prior to their movement through the vessel wall and into surrounding tissue. For instance, certain inflammation-triggering compounds such as bacterial endotoxin, tumor necrosis factor, and interleukin 1 act directly on the vascular endothelium to promote adhesion of leukocytes and lymphocytes. These cells then move through the blood vessel wall and into areas of injury or infection.

Cellular adhesion to vascular endothelium is also thought to be involved in tumor metastasis, circulating cancer cells apparently take advantage of the body's normal inflammatory mechanisms and bind to areas of blood vessel walls where the endothelium is activated.

Recent work has revealed that a specialized cell 5 surface receptor on activated endothelial cells (designated ELAM-1) is involved in the recognition of various circulating cells by the endothelium. This receptor is a surface glycoprotein with a lectin-like domain, a region with homology to epidermal growth factor, and a region with homology to complement regulatory proteins (see. Bevilacqua et al., Science 243:1160 (1989), which is incorporated herein by reference). ELAM-1 has been shown to mediate endothelial leukocyte adhesion, which is the first step in many inflammatory responses. Specifically, ELAM-1 binds human neutrophils, lymphocytes (e.g., NK cells), monocytes, and the promyelocytic cell line HL-60.

Cell surface receptors of this general class are expressed in a variety of cells. For example, GMP-140 is a related receptor present on the surface of platelets, where it mediates platelet-leukocyte interactions, MEL-14 is a cell surface receptor of lymphocytes, and acts as a lymph node homing receptor. The exact nature of the ligand recognized by these receptors, however, has remained largely unknown. Bevilacqua has suggested the term selectin'* for this class of receptors because of their lectin-like domain and the selective nature of their adhesive functions.

Various methods have been previously developed to block the action of these receptors and thus inhibit cellular adhesion. For instance, the use of monoclonal antibodies directed to ELAM-1 has been proposed as a method to inhibit endothelial-leukocyte adhesion as a treatment for pathological responses, such as inflammation. Endothelial interleukin-8 has also been shown to be an inhibitor of leukocyte-endothelial interactions. To date, however, insufficient understanding of the interaction of the ligand and receptor molecules on the respective cells has hindered efforts to produce highly specific, efficient inhibitors of selectin-mediated cellular adhesion useful in therapeutic regimens.

SUMMARY OF THE INVENTION The present invention relates to methods, for screening for therapeutically effective blocking agents capable 5 of inhibiting fucosyltransferase activity. Samples suspected of containing the blocking agents of the present invention are typically assayed for inhibition of in vitro cellular adhesion mediated by a selectin receptor, other suitable assays include assays for inhibition of fucosyltransferase activity or for inhibition of inflammatory diseases in mammals.

The sample is usually obtained from synthetically produced compounds or from natural sources, such as a cell (e.g., microbes or higher organisms) culture broth. The assays additionally include steps of fractionating and subfractionating samples to isolate the components of the sample responsible for the inhibitory activity. Thus, a substantially pure blocking agent may be obtained.

The present invention also provides methods of inhibiting intercellular adhesion mediated by a selectin cell surface receptor in a mammal. These methods entail the administration to the mammal of a therapeutically effective dose of a blocking agent capable of inhibiting fucosyltransferase activity. Preferred target enzymes are al,3 fucosyltransferases, in particular, those that fucosylate a sialylated N-acetyllactosamine moiety. The resulting fucosylated oligosaccharide is designated here as SLX.

SLX is typically on a cell surface glycoconjugate, usually a glycoprotein or glycolipid. The glycoconjugate may be expressed on the surface of a leukocyte. The selectin cell surface receptor that recognizes the SLX ligand is usually expressed on vascular endothelial cells and is typically ELAM-1.

Exemplary blocking agents of the present invention are unreactive sugar substrates, such as unreactive sugar nucleotides or unreactive acceptor substrates. Acceptor substrates are typically synthesized according to the methods described herein.

IE 91265G-------4 The present invention further provides pharmaceutical compositions comprising a suitable carrier and one or more blocking agents. The pharmaceutical compositions are useful for treating a variety of inflammatory disease process mediated by selectin cell surface receptors, such as psoriasis reperfusion injury, and rheumatoid arthritis.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Compositions and methods are provided for inhibiting 10 inflammatory and other disease responses mediated by cellular adhesion. Specifically, the present invention relates to blocking agents which have the ability to inhibit the activity of fucosyltransferases involved in the synthesis of ligands recognized by selectin cell surface receptors. Preferred blocking agents specifically inhibit the enzyme responsible for fucosylating a sialylated N-acetyllactosamine moiety. Methods are also disclosed for preparing the blocking agents as well as various screening assays to identify suitable candidates. In addition, the present invention is directed to screening assays for identifying blocking agents from natural sources such as microbial broths. Therapeutic and other uses for these compounds are also provided.

The present invention is directed to inhibition of the synthesis of the carbohydrate moiety recognized by selectin cell surface receptors. Selectin receptors comprise a lectinlike domain, which likely determines their specificity. See, e.g.. Bevilacqua et al., Science, supra♦ Evidence indicates that a sialylated, fucosylated N-acetyllactosamine unit, designated here as SLX, is a moiety recognized by the lectin region of the selectin receptor. See, copending and commonly assigned U.S. patent application Serial No. 538,853, filed June, 15, 1990, which is incorporated by reference. SLX has the following structure: NeuAcd2,3Gal01,4GlcNAc01 |el,3 Fuc It has been demonstrated that mutant CHO cells bearing the SLX antigen bind to activated human vascular endothelial cells. See, U.S. Serial No. 538,853, supra. Neither wild type CHO cells nor other related glycosylation mutant CHO cell lines without SLX showed the same level of binding. In particular, it was demonstrated that the presence of both the fucosyl residue in al,3 linkage to the Nacetylglucosamine and the sialic acid in a2,3 linkage to the galactose are likely necessary for the recognition of this moiety by the selectin receptor.

The nomenclature used to describe oligosaccharides follows conventional nomenclature. Standard abbreviations for individual monosaccharides are used. For instance, 2-N-acetylglucosamine is represented by GlcNAc, -N-acetylneuraminic acid (a sialic acid) is NeuAc, fucose is Fuc, galactose is Gal, and glucose is Glc. Unless otherwise indicated, all sugars are D-isomers in the cyclic configuration. Fucose, however, is typically present as the Lisoaer. The two anomers of the cyclic forms are represented by a and β.

The monosaccharides are generally linked by glycosidic bonds to form oligo- and polysaccharides. The orientation of the bond with respect to the plane of the rings is indicated by β and β. The particular carbon atoms that form the bond between the two monosaccharides are also noted. Thus, a β glycosidic bond between C-1 of galactose and C-4 of glucose is represented by Gal01,4Glc. For the D-sugars (e.g. D-GlcNAc, D-Gal, and D-NeuAc) the designation a means the oxygen atom attached to C-1 (C-2 in NeuAc) is below the plane of the ring and β is above the ring. In the case of L-fucose, the a designation means the oxygen atom is above the ring and β means it is below.

The invention described herein is based in part on the recognition that leukocyte adhesion to ELAM-1 on activated endothelial cells is prevented by blocking the biosynthesis and cell surface expression of the SLX ligand. The biosynthesis of the SLX ligand as a terminal sequence on glycoprotein and glycolipid carbohydrate groups occurs entirely in the Golgi apparatus, a subcellular compartment of the secretory pathway that contains glycosyltransferases. Inhibitors of either fucosyltransferases or sialyltransferases prevent the synthesis of the SLX ligand. However, generalized inhibition of the sialytransferase, a common enzyme involved in the synthesis of many glycoproteins, would likely expose underlying carbohydrate groups and would ‘activate· biological activities normally masked by sialic acid. (For a review, see Schauer, Adv. Carbohvdr. Chem. Biochem.. 40:131-234 (1982), which is incorporated herein by reference.) No such complication exists with inhibition of fucosyltransferase activity.

The preferred fucosyltransferase inhibitors of the present invention have the ability to cross the cell membrane and enter the golgi apparatus, the site of SLX synthesis.

Thus, the blocking agents are preferably sufficiently , hydrophobic to allow diffusion through the membrane.

Generally, they have no other adverse effects on cellular metabolism, so that other glycosylation reactions proceed while specific fucosylation is inhibited. The blocking agents are preferably relatively small molecules, thereby avoiding immunogenicity and allowing passage through the cell membrane.

Ideally, they have a molecular weight of between about 500-2000 daltons, but may have molecular weights up to 5000 or more, depending upon the desired application.

The inhibitors of the present invention preferably have strong affinity for the target enzyme, so that at least about 70% inhibition of fucosyltransferase activity is achieved, more preferably about 75%-85% and most preferably 90%-95% or more. The affinity of the enzyme for the inhibitor : is preferably sufficiently strong that the dissociation constant, or K , of the enzyme-inhibitor complex is less than about 10”5 M, typically between about io'6 and 10’’ M.

In addition to generalized fucosyltransferase inhibitors, specific inhibitors of individual fucosyltransferases can be used. While many cells produce carbohydrate groups that contain fucose, only a limited number of cell types, including neutrophils, monocytes and certain lymphocytes, express the specific fucosyltransferase involved in the synthesis and cell surface expression of the SLX ligand. Macher, et al. Leukemia Res. 14:119-130 (1990) which is incorporated herein by reference. Consequently, fucosyltransferase inhibitors directed at the specific fucosyltransferase involved in the synthesis of the SLX ligand effect the carbohydrate groups of a limited number of cell types.

I. Glvcosvltransferases and the synthesis of glycosides The biology and biochemistry of the enzymes involved in the synthesis of specific glycosides such as SLX have been extensively studied. Thus, many methods for inhibiting fucosyltransferases will be readily apparent to those skilled in the art. Glycosyltransferases, the general group of enzymes that catalyze the synthesis of these moieties, catalyze the transfer of a monosaccharide from a glycosylnucleotide, the donor substrate, to an acceptor substrate. The acceptor substrate may be another glycosyl residue, a polypeptide, or a lipid, depending on the specificity of the transferase. See, generally. Beyer et al., Adv, in Enzvm. 52:24 (1981), which is incorporated herein by reference.

Glycosyltransferases are grouped into families based on the type of sugar residue transferred. For example, enzymes that transfer sialic acid are called sialytransferases, those that transfer fucose are called fucosyltransferases, and those that transfer galactose are called galactosyltransferases. In each family there are typically 10-15 different enzymes required to elaborate the diverse carbohydrate structures found on glycoproteins and glycolipids of animal cells. Each enzyme makes a defined structure based on the donor and acceptor substrates they utilize, and the anomeric linkage formed in the transfer reaction.

The synthesis of the SLX ligand occurs through an ordered addition of sialic acid and fucose through the concerted action of a specific sialytransferase and a specific fucosyltransferase: Ga01,4GlcNAc-R CMP-NeuAc ) CMP S ialytrans ferase NeuAca2,3Gal01,4GlcNAc-R GDP-Fuc.

Fucosyltransferase GDP’ NeuAca2,3 Gal01,4GlcNAc~R SLX ligand Fucal,3 Fucosyltransferases and sialytransferases may be further classified by the glycosidic linkages they produce. While several fucosyltransferases produce the Fucal,3GlcNAc linkage and several sialytransferases produce the NeuAca2,3Gal linkage, not all are able to participate in the synthesis of the SLX ligand (see Beyer et al. supra: Campbell et al., J, Biol.

Chem.. 259:11208 (1984); Stanley and Atkinson, J. Biol. Chem, 264:11374 (1988) all of which are incorporated herein by reference).

The fucosyltransferase associated with the synthesis of the SLX ligand recognized by selectins adds a fucose to a sialylated N-acetyllactosamine moiety as shown above. An enzyme catalyzing this reaction in CHO cells has been identified and characterized. See. Howard et al., J, Biol.

Chem.. 262:16830 (1987) which is incorporated herein by reference, and Campbell et al., supra. Other workers have purified from human milk the enzyme responsible for the cl, 3 linkage of fucose to N-actyellactosamine. See. Prieels et al., J, Biol, Chem.. 256:10456 (1981), which is incorporated herein by reference. Fucosyltransferases have also been purified from a variety of other sources. Examples include human parotid saliva (Tamagawa et al., J. Dent, Res.. 66:756-760 (1987)), « submaxillary glands (Beyer et al., J. Biol. Chem.. 255:53735379 (1980)), serum (Madiyalakan et al., An^l, Bioch.. 152:2228 (1986)), bone marrow (Pacuszka, et al., FEBS Lett.. 41:348IE 912650 351 (1974)), amniotic fluid (Mitsakos et al., Hooe-Sevler Biol. Chem.. 370:239-243 (1989)), and human lung cancer cells (Holmes et al., J. Biol. Chem.. 260:7619-7627)). All of the above references are incorporated herein by reference.

II. Inhibition of enzyme activity Having identified the target enzyme to be inhibited (i.e., fucosyltransferase), many approaches can be used to block its activity. Enzyme inhibition generally involves the interaction of a substance with an enzyme so as to decrease the rate of the reaction catalyzed by that enzyme. Inhibitors can be classified according a number of criteria. For example, they may be reversible or irreversible. An irreversible inhibitor dissociates very slowly, if at all, from its target enzyme because it becomes very tightly bound to.the enzyme, either covalently or noncovalently. Reversible inhibition, in contrast, involves an enzyme-inhibitor complex which may dissociate.

Inhibitors can also be classified according to whether they are competitive, noncompetitive or uncompetitive inhibitors. In competitive inhibition for kinetically simple systems involving a single substrate, the enzyme can bind either the substrate or the inhibitor, but not both.

Typically, competitive inhibitors resemble the substrate or the product(s) and bind the active site of the enzyme, thus blocking the substrate from binding the active site. A competitive inhibitor diminishes the rate of catalysis by effectively reducing the affinity of the substrate for the enzyme. Typically, an enzyme may be competitively inhibited by its own product because of equilibrium considerations. Since the enzyme is a catalyst, it is in principle capable of accelerating a reaction in the forward or reverse direction.

Noncompetitive inhibitors allow the enzyme to bind the substrate at the same time it binds the inhibitor. A noncompetitive inhibitor acts by decreasing the turnover number of an enzyme rather than diminishing the proportion of free enzyme. Another possible category of inhibition is mixed or uncompetitive inhibition, in which the inhibitor affects the binding site and also alters the turnover number of the enzyme.

Enzyme inhibition of kinetically complex systems involving more than one substrate, as is the case for glycosyltransferases, are described in Segel, Enzyme Kinetics. (Wiley, N.Y. 1975), which is incorporated herein by reference.

Numerous ways of inhibiting enzyme activity are well known in the art. Examples of agents capable of inhibiting enzyme activity include, immunoglobulins, suicide substrates, alkylating agents, and various substrate analogs. For a review, see Fersht, Enzyme Structure and Mechanism (2d ed. 1985), which is incorporated herein by reference.

III. Inhibitors of fucosyltransferase activity A. Sugar nucleotides Methods for inhibiting glycosyltransferase activities, including fucosyltransferase activities, are also well known. As discussed above, the donor substrate of glycosyltransferases are sugar nucleotides, usually diphosphonucleosides. For example, uridine diphosphosugars are donor substrates for the formation of glycosides of glucose, galactose, N-acetylglucosamine, xylose, and glucuronic acid.

Guanosine diphosphosugars are donor substrates for the synthesis of glycosides of mannose and fucose. The glycosides of the sialic acids are formed by transfer from cytidine monophosphosialic acid.

Using this knowledge, one of skill in the art can readily synthesize a number of sugar nucleotides which can then be tested to identify those capable of maximum inhibition of a specific enzyme. The term sugar nucleotide as used herein refers both to sugar nucleotides discussed above and to various analogs thereof that might be synthesized or isolated from natural sources. The number of variations on this structure is limitless. For instance, both the ester linkage between the sugar and phosphate and the anhydride linkage of the pyrophosphate are potential targets of enzymatic cleavage. Replacement of the 0-P or C-0 linkage with a more stable C-P bond provides nucleotidediphosphate sugar analogs that are more resistant to enzymatic degradation. Such compounds have the potential to selectively inhibit glycoprotein or glycolipid synthesis by acting as substrate analogs of a particular glycosyltransferase. See, e.g, . Vaghefi, et al., J. Med. Chem, 30:1383-1391 (1987), and Vaghefi, et al., J, Med, Chem. 30:1391-1399 (1987), both of which are incorporated herein by reference.

Another approach is to replace the diphosphate bridge between the sugar residue and the nucleoside moiety. For instance, the diphosphate bridge can be replaced with an isosteric -0C0NHS02O~ residue. See, Camarasa, et al., J, Med, Chem. 28:40-46 (1985), which is incorporated herein by reference.

Analogs of sugar nucleotide capable of inhibiting glycosylation have been used as antibiotics and antiviral agents. Examples of such compounds include 2-deoxy-D-glucose, which is transformed to either UDP-2dGlc or GDP-2dGlc and in that form inhibits glycosylation of glycoproteins in the viral envelope. DeClercq, Biochem, J, 205:1 (1982) which is incorporated herein by reference. Antibiotics such as tunicamycin and streptovirudin are also effective because of their ability to inhibit glycosylation. For instance, tunicamycin is an analog of UDP-GlcNAc, the donor substrate for N-acetylglucosaminyltransferases. The replacement of diphosphate bridge with a carbon chain allows tunicamycin to cross the cell membrane but still readily bind the active site of the enzyme. The structure of these and related compounds provide one of skill in the art with direction in designing and synthesizing compounds with similar inhibitory effects in accordance with the present invention as described herein.

As discussed above, nucleotides are the byproduct of the reaction by which glycosyl residues are transferred to acceptor substrates. Nucleotides have been found to competitively inhibit glycosyltransferase. Thus, various nucleotides and their analogs have potential as inhibitors of these enzymes. For instance, al,3/l,4 fucosyltranferase activity from human milk is inhibited by GDP and GMP. Prieels, et al., J, Biol. Chem. 256:10456-10463 (1981) which is incorporated herein by reference. The same compounds have shown similar inhibitory effect on fucosyltransferase isolated from mutant CHO cells (Campbell, et al., supra and el,2 fucosyltransferase from rat small Intestines (Bella, et al., Biochem J.. 125:1157-1158 (1971)).

B. Acceptor substrate analogs In addition to the donor substrate analogs, analogs 5 of acceptor substrates may also be used as inhibitors. Again, the skilled artisan will recognize a variety of possible structures that can be used. Because of the acceptor substrate specificity of fucosyltransferases, specific inhibition of the fucosyltransferase that recognizes the sialylated N10 acetyllactosamine moiety important in cell adhesion can be obtained. Ideally, the inhibitory compounds should be capable of acting as specific acceptor substrates for a given enzyme, even in the presence of other enzymes. In addition, the compound should be an efficient acceptor substrate. Thus, the KA of the inhibitor should be at least about IO'5 M, more preferably at least about IO'7 M.

Preferred acceptor substrate analogs of the present invention bind very tightly to the enzyme, but cannot be fucosylated. Examples of such compounds include: wherein Y=H and X~H, F, SH, OMe, SHE, NH2, NHAc, and CH2SH. Alternatively, X=H, and Y is the substituents listed for X, above. R can be 0 Alkyl, O Alkylaryl, S Alkyl, or S Aryl.

These compounds can be synthesized enzymatically 25 starting with substituted N-acetylglueosamine. Galactose is added to C-4 of the N-acetylglucosamine using β1,4 galactosyltransferase and UDP-Gal. Next, sialic acid is added to C-3 of galactose using a2,3 sialyltransferase and CMP-SA. Alkaline phosphatase, such as calf intestine alkaline phosphatase (CIAP), is typically included in each step to consume the nucleoside phosphate byproduct which may inhibit the reaction.

In addition, the compounds can be synthetically produced using methods well known in the art according to the scheme below: ▼ (Π) Ο 2.3-SA tranaferaae CMP-SA CUP The above reactions begin by reversibly protecting the hydroxyl groups on C-4 and C-6 of N-acetylglucosamine so that the X and Y substitution on C-3 can be accomplished, the resulting compound is Labeled (I). Next, C-3, C-4, and C-6 on a galactal are protected, the resulting compound is labelled (II). (I) and (II) are then coupled, followed by a deprotection step to restore the hydroxyl groups. The final step is the enzymatic transfer of N-acetylneuraminic acid (a sialic acid) to C-3 of the galactose. The product is NeuAcc2,3galj91,4GlcNAc, wherein the C-3 of the N-acetylglucosamine residue is variously substituted as described above. Most of the chemistry involved in the synthesis follows techniques well known to the skilled artisan. The synthesis of (II) and the coupling of (I) and (II) are as described in Halcomb et al., J.· Am. Chem, Soc.. 111:7638-7640 (1984) and Friesen et al., J. Am. Chem. Soc.. 111:6656-6660 (1989), both of which are incorporated herein by reference.

Another approach is to use various glycoconjugates or oligosaccharides bearing unfucosylated SLX as competitive inhibitors of the enzyme. The resulting fucosylated compound would additionally be recognized by, and block, the corresponding selectin receptor, thus leading to further inhibition of cellular adhesion. For instance, it has been demonstrated that various glycosphingolipids such as lactoneotetraosylceramide act as acceptors for both al,2 and al,3 fucosyltransferases from human serum. See. Pacuszka, et al., European J, Biochem, 64:499-506 (1976), which is incorporated herein by reference. In addition, fucosyltransferases isolated from human carcinomas fucosylate other glycosphingolipids such as sialosyl 2,6 ! lactonorhexaosylceramide. Holmes, et al., J, Biol, Chem, 260:7619-7627 (1985), which is incorporated herein by reference.

Specific synthetic acceptors can also be used. For instance, N-acetyl-2'-O-methyllactosamine has been shown to be an efficient acceptor of al,3 fucosyltransferase from human serum. Madiyalakan, et al., Anal. Biol, Chem. 152:22-28 (1986), which is incorporated herein by reference. Other possible acceptors include lacto-N-fucopentaose I or 2' fucosyllactose. Tamagawa, et al., J. Dent. Res. 66:756-760 (1987), which is incorporated herein by reference.

The specificity of glycosyltransferases most likely 5 arises from their ability to recognize both the acceptor substrate and the sugar nucleotide donor. Thus, glycosyltransferase inhibitors can also be synthesized which contain structural elements of both the donor and acceptor substrates. See, Palcic, et al., J, Biol. Chem, 264:1717410 17181 (1989), which is incorporated herein by reference.

C. Additional inhibitors Naturally occurring molecules which show inhibitory effects may also be isolated for use in the present invention. The biosynthesis of glycoproteins or glycolipids is a complex metabolic pathway that depends on many factors for regulation. Naturally occurring inhibitory compounds can be purified and used to further inhibit activity. For instance, the presence of an endogenous cytosolic protein inhibitor of intestinal fucosyltransferase activity has been shown. Martin, et al., Biochem. Biophvs. Res. Comm. 166:1024-1031 (1990), which is incorporated herein by reference.

Yet another tactic to inhibit fucosyltransferase activity is to use immunoglobulin molecules raised against the particular enzyme of interest. See, e.g.. White et al., Biochem.. 29:2740-2747 (1990). Thus, the multitude of techniques available to those skilled in the art for production and manipulation of various immunoglobulin molecules can be applied to inhibit intercellular adhesion. The immunoglobulins may exist in a variety of forms besides antibodies, including for example, Fv, Fab, and F(ab)2, as well as in single chains fe.q.. Huston et al., Proc. Nat. Acad, Sci. U.S.A. 85:5879-5883 (1000) and Bird et al., Ceiewee 242:423-426 (1900), which are incorporated herein by reference). (gee. generally. Hood et al., Immunology, 2nd ed., Benjamin, N.Y. (1984), and Hunkapiller and Hood, Nature 323:15-16 (1986), which are incorporated herein by reference.) Antibodies which bind the enzyme may be produced by a variety of means. The production of non-human monoclonal antibodies, e.g.. murine, lagomorpha, equine, etc., is well known and may be accomplished by, for example, immunizing the animal with fucosyltransferase or a fragment thereof conjugated to a carrier. Antibody-producing cells obtained from the immunized animals are immortalized and screened, or screened first for the production of antibody which inhibits the interaction of the enzyme with the substrate and then immortalized. For a discussion of general procedures of monoclonal antibody production, see. Harlow and Lane, Antibodies, A Laboratory Manual (1988), which is incorporated herein by reference.

Antisense technology may also be used to inhibit fucosyltransferase activity. Generally, this approach involves introducing into a cell a sequence of DNA or RNA that is complementary to mRNA encoding a particular protein. The introduced nucleic acid sequence binds the mRNA and prevents translation of the encoded polypeptide. Antisense RNA has been shown to regulate gene expression in mammalian cells. For a general discussion of antisense technology, see, Antisense DNA and RNA. (Cold Spring Harbor Laboratory, D. Melton, ed., 1988) which is incorporated herein by reference.

IV. Screening methods for, identifying blocking agents In light of the present disclosure, it is now feasible to identify therapeutically effective blocking agents by screening a variety of compounds and mixtures of compounds for their ability to inhibit fucosyltransferase activity. The use of screening assays to discover naturally occurring compounds with desired activities is well known and has been widely used for many years. For instance, many compounds with antibiotic activity were originally identified using this approach. Examples of such compounds include monobactams and aminoglycoside antibiotics. Compounds which inhibit various enzyme activities have also been found by this technique, for example, mevinolin, lovastatin, and mevacor, which are inhibitors of hydroxymethylglutamyl Coenzyme A reductase, an enzyme involved in cholesterol synthesis. Antibiotics that inhibit glycosyltransferase activities, such as tunicamycin and streptovirudin have also been identified in this manner.

Thus, another important aspect of the present invention is directed to methods for screening samples for inhibiting activity. A sample’1 as used herein may be any mixture of compounds suitable for testing in a fucosyltransferase activity. A typical sample comprises a mixture of synthetically produced compounds or alternatively a naturally occurring mixture, such as a cell culture broth. Suitable cells include any cultured cells such as mammalian, insect, microbial or plant cells. Microbial cell cultures are composed of any microscopic organism such as bacteria, protozoa, yoact, fungi and the like. culture broths are screened according to standard techniques. See. e.g., Meyer et al., Can, J. of Microbiol.. 25:1232-1238 (1979), Catino et al., Cancer chemother Pharmacol. :240-243 (1985), and Mirabelli et al., J. Antibiotics, 38:758-766 (1985), all of which are incorporated herein by reference. i A broth is typically produced by spinning down the cultured cells and collecting the supernatant. Alternatively, the cells are homogenized using techniques well known in the art to obtain a more complex mixture in the supernatant. Synthetically produced compounds are derived using standard chemical synthesis techniques well known in the art.

In the typical screening assay, a sample, such as a fungal broth, is added to a standard fucosyltransferase assay. If inhibition of activity as compared to control assays is found, the mixture is usually fractionated to identify components of the sample providing the inhibiting activity.

The sample is fractionated using standard methods such as ion exchange chromatography, affinity chromatography, electrophoresis, ultra filtration, HPLC and the like. See. e.g.. Protein Purification, Principles and Practice. (SpringerVerlag, 1982), which is incorporated herein by reference. Each isolated fraction is then tested for inhibitory activity. If - 35 desired, the fractions are then further subfractionated and tested. This subfractionation and testing procedure can be repeated as many times as desired.

By combining various standard purification methods, a substantially pure compound suitable for in vivo therapeutic testing can be obtained. A substantially pure blocking agent as defined herein is an inhibitory compound which migrates largely as a single band under standard electrophoretic conditions or largely as a single peak when monitored on a chromatographic column. More specifically, compositions of substantially pure blocking agents will comprise less than ten percent miscellaneous compounds.

Fucosyltransferase activity and its inhibition is typically assayed according to standard methods for determining enzyme activity. For a general discussion of enzyme assays, see, Rossomando, Measurement of Enzyme Activity in Guide to Protein Purification, Vol. 182, Methods in Enzymology (Deutscher ed., 1990) which is incorporated herein by reference, and Fersht, supra.

An assay for fucosyltransferase activity typically contains a buffered solution adjusted to physiological pH, a source of divalent cations, a donor substrate (usually labelled guanosine diphosphofucose), an acceptor substrate (e.g., afucosyl SLX), fucosyltransferase (typically al,3 fucosyl transferase from LEC ii cells, HL-60 cells, HT-29 cells, certain adenocarcinomas, or polymorphonuclear leukocytes), and the sample or fraction of a sample whose inhibitory activity is to be tested. After a predetermined time at 23 *C or 37*C, the reaction is stopped and the fucosylated product is isolated and measured according to standard methods (e.g., in a scintillation counter). Inhibition of fucosyltransferase activity in an assay as defined herein refers to a decrease in enzyme specific activity of at least about 70%, more preferably at least about 90%. Examples of particular assays that may be used ore found in the following referencea i Tanagowu et al. , suprai Prieels et al., supra? Holmes, et al., supra; Mitsakos et al., supra? and Madiyalakan, supra.

After a fraction of a sample is identified that exhibits good inhibiting activity, its ability to inhibit in vitro cellular adhesion mediated by a selection is tested.

This can be carried out according to the assay described in U. S. Serial No. 538,853, supra. For example, the leukocyte cell line HL-60 can be grown in the presence of 10‘6 to 10’3 molar blocking agent to prevent the synthesis of fucosylated carbohydrate groups on the surface of the cells. These cells can then be used to test for the effectiveness of the agent in blocking cell adhesion. Inhibition of An vitro cellular adhesion as used herein refers to the ability to prevent adhesion of at least about 70% of an appropriate cell bearing the six ligand to activated endothelial cells, more preferably about 90%.

Finally, samples and fractions of samples can be further tested for their ability to inhibit inflammatory disease responses in laboratory animals. Animals can be treated with pharmalogieal doses of the blocking agent to block addition of fucose to cell surface carbohydrates of leukocytes. Animals can then be tested for susceptibility for neutrophil infiltration into sites of inflammation (Mourshargh, S. et al.

J. Immunol. 142:3193-98 (1989), which is incorporated herein by 1 reference), reperfusion injury (Vedder, N.B. et al., Proc. Nat. 20 Acad. Sci. USA 87:2643-46, which is incorporated herein by reference) and other animal models suitable for assay of leukocyte induced tissue damage.

V. Uses for blocking agents of the present invention The blocking agents of the present invention have a variety of applications. In recent years, the study of fucosyltransferases has grown as a result of the recognition of their biological importance. For instance, these enzymes are known to be involved in the synthesis of Lewis blood group antigens Le* and Leb, which include fucosyl residues. Prieels et al., supra. Elevations in fucosyltransferase activities have also been reported in cancer cells (Holmes et al., supra). suggesting that fucosyltransferases may be markers for early diagnosis of malignancies or for monitoring disease status during chemotherapy. For instance, the blocking agents which act as substrate analogs are used for in vitro diagnosis of cells, (e.g., cancer cells) which express the particular fucosyltransferase of interest. The response of the cells to a biologically effective dose of the agent can then be determined. A biologically effective dose as used herein refers to an amount sufficient to produce a biological effect detectable either in an in vitro test or in vivo applications, such as therapeutic uses. Thus, the blocking agents of the present invention are especially useful in various in vitro studies that help elucidate the activities and specificities of fucosyltransferases from different sources, such as human cancer tissue. See, e.g. Holmes, supra. and Price, supra.

The compositions of the present invention are also used therapeutically to selectively inhibit fucosyltransferase activity associated with a variety of disorders. For instance, a number of inflammatory disorders are associated with selectins expressed on vascular endothelial cells. The term inflammation is used here to refer to reactions of both the specific and non-specific defense systems. A specific defense system reaction is a specific immune system reaction to an antigen. Example of specific defense system reactions include antibody response to antigens, such as viruses, and delayedtype hypersensitivity. A non-specific defense system reaction is an inflammatory response mediated by leukocytes generally incapable of immunological memory. Such cells include granulocytes, macrophages, and neutrophils. Examples of nonspecific reactions include the immediate swelling after a bee sting, and the collection PMN leukocytes at sites of bacterial infection (e.g., pulmonary infiltrates in bacterial pneumonias and pus formation in abscesses).

Other disorders treatable by compositions of the present invention include, e.g., rheumatoid arthritis, postischemic leukocyte-mediated tissue damage (reperfusion injury), acute leukocyte-mediated lung injury (e.g., adult respiratory distress syndrome), septic shock, and acute and chronic inflammation, including atopic dermatitis and psoriasis. In the case of reperfusion injury, the blocking agents are ideally used prophylactically prior to heart surgery to enhance postsurgical recovery. In addition, tumor metastasis can be prevented by inhibiting the adhesion of circulating cancer cells. Examples include carcinoma of the colon and melanoma.

By way of example, psoriasis is particularly amenable to treatment by compositions of the present invention’. The treatment may be initiated prophylactically or after the onset of a psoriatic episode.

The dose of the blocking agents of the invention for treatment of inflammatory disease will vary according to, e.g., the blocking agent, the manner of administration, the particular disease being treated and its severity, the overall health and condition of the patient, and the judgment of the prescribing physician. For example, for the treatment of psoriasis, the dose is in the range of about 50 pg to 2,ooo mg/day for a 70 kg patient. Ideally, therapeutic administration should begin as soon as possible after the onset of the psoriatic episode.

The pharmaceutical compositions are intended for parenteral, topical, oral or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment. For the treatment of psoriasis, the pharmaceutical compositions are preferably administered topically.

For topical application, non-sprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water are typically used. Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.

For aerosol administration, the compounds are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of blocking agents are 0.l%-10% by weight, preferably l%-5%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride such as, for example, ethylene glycol, glycerol, erythritol, arbitol, mannitol, sorbitol, the hexitol anhydrides derived from sorbitol, and the polyoxyethylene and polyoxypropylene derivatives of these esters. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-20% by weight of the composition, preferably ( 0.25-5%. The balance of the composition is ordinarily propellant. Liquefied propellants are typically gases at ambient conditions, and are condensed under pressure. Among suitable liquefied propellants are the lower alkanes containing up to 5 carbons, such as butane and propane; and preferably fluorinated or fluorochlorinated alkanes. Mixtures of the above may also be employed. In producing the aerosol, a container equipped with a suitable valve is filled with the appropriate propellant, containing the finely divided compounds and surfactant. The ingredients are thus maintained at an elevated pressure until released by action of the valve.

This invention also provides compositions for intravenous administration which comprise a solution of the blocking agent dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

The concentration of the blocking agent which may be combined to form a blocking agent cocktail under certain circumstances for increased efficacy in the pharmaceutical formulations can vary widely, i.e., from less than about 0.05% ' usually at or at least about 1% to as much as io to 70% by weight and will be selected primarily by fluid volume», viscosities, etc., in accordance with the particular mode of administration selected.

Thus, a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 mi of sterile Ringer's solution, and a unit dosage comprising 2-2,000 mg of the compound. Actual methods for preparing parenterally administrable compounds will be known or apparent to those skilled in the art and are described in more detail in for examp1e, Remington's Pharmaceutical Science, 171h ,ed., Mack publishing Company, Easton, PA (1985), which is incorporated herein by reference. For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more blocking agent of the invention, preferably 15%.

The compositions containing the compounds can be administered for prophylactic and/or therapeutic treatments.

In therapeutic applications, compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as therapeutically effective dose. Unit dosages effective for this use will depend on the severity of the disease and the weight and general state of the patient, but generally range from about 0.5 mg to about 2,000 mg of blocking agent per day for a 70 kg patient, with dosages of from about 25 mg to about 250 mg of the compounds per day being more commonly used.

In prophylactic applications, compositions containing the compounds of the invention are administered to a patient susceptible to or otherwise at risk of a particular disease. Such an amount is defined to be a prophylactically effective dose. In this use, the precise amounts again depend on the patient's state of health and weight, but generally range from about 0.5 mg to about 2,000 mg per 70 kilogram patient, more commonly from about 25 mg to about 250 mg per 70 kg of body weight.

Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of inhibitor of this invention sufficient to effectively treat the patient.

Having now fully described this invention, it will be apparent to one of ordinary skill in the art that many,changes and modifications can be made thereto without departing from the spirit or scope of invention set forth herein.

Claims (33)

WHAT IS CLAIMED IS:

1. A method for identifying a therapeutically effective blocking agent in a sample, which blocking agent is 5 capable of inhibiting cellular adhesion mediated by a selectin cell surface receptor, the method comprising the step of assaying the sample for the ability to inhibit in vitro cellular adhesion by inhibiting fucosyltransferase activity. 10

2. A method of claim 1 further comprising the step of assaying the sample for inhibition of fucosyltransferase activity in a fucosyltransferase assay.

3. A method of claim 1 further comprising the step 15 of testing the sample for inhibition of an inflammatory disease process mediated by a selectin cell surface receptor in a mammal.

4. A method of claim 1 wherein the sample is a cell 20 culture broth.

5. A method of claim 4 wherein the cell culture is a microbial culture. 25

6. A method for identifying a therapeutically effective blocking agent capable of inhibiting cellular adhesion mediated by a selectin cell surface receptor, the method comprising the steps of; a. assaying a cell culture broth for 30 inhibition of fucosyltransferase activity in a fucosyltransferase assay; b. assaying fractions of an inhibiting broth from step (a) for inhibition of fucosyltransferase activity in a fucosyltransferase assay; and c. assaying positive fractions from step (b) for in vitro inhibition of cellular adhesion.

7. A method of claim 6 further comprising the step of testing the positive fractions for inhibition of an inflammatory disease process mediated by a selectin cell surface receptor in a mammal.

8. A method of claim 7 wherein the cell culture is a microbial culture.

9. A method of claim 6 further comprising the steps 10. Of: subfractionating a positive fraction from step (b) and assaying the subfractions for inhibition of fucosyltransferase activity in a fucosyltransferase assay; and repeating the subfractionating step until a 15 substantially pure blocking agent is obtained.

10. A compound identified by the method of claims 1, or 6. 20

11. A method of inhibiting intercellular adhesion mediated by a mammalian selectin cell surface receptor, the method comprising contacting the receptor with a biologically effective dose of a blocking agent capable of inhibiting fucosyltransferase activity.

12. A method of claim 11 wherein the fucosyltransferase is el,3 fucosyltransferase.

13. A method of claim 12 wherein the al,3 30 fucosyltransferase fucosylates a sialylated N-acetyllactosamine moiety.

14. A method of claim 11 wherein the fucosyltransferase fucosylates an oligosaccharide moiety on a 35 gly coconjugate expressed on a cell surface.

15. A method of claim 14 wherein the glycoconjugate is a glycoprotein or glycolipid.

16. A method of claim 14 wherein the glycoconjugate is on a leukocyte. 5

17. A method of claim 11 wherein the selectin cell surface receptor is expressed on vascular endothelial cells.

18. A method of claim 18 wherein the selectin cell surface receptor is ELAM-1.

19. A method of claim 11 wherein the blocking agent is an unreactive substrate of fucosyltransferase.

20. A method of claim 20 wherein the blocking agent 15 is an unreactive sugar nucleotide.

21. A method of claim 11 wherein the blocking agent is an unreactive acceptor substrate of fucosyltransferase. I 20

22. A pharmaceutical composition comprising a suitable carrier and a blocking agent in a unit dosage capable of inhibiting fucosyltransferase activity in a mammal.

23. A pharmaceutical composition of claim 22 wherein 25 the blocking agent is an unreactive sugar substrate.

24. A method of claim 23 wherein the blocking agent is an unreactive sugar nucleotide. 30

25. A pharmaceutical composition of claim 23 wherein the blocking agent is an unreactive acceptor substrate.

26. A method of treating an inflammatory disease process mediated by a selectin cell surface receptor in a 35 mammal, the method comprising administering to the mammal a therapeutically effective dose of the pharmaceutical composition of claim 22.

27. A method of claim 26 wherein the selectin cell surface receptor is expressed by a vascular endothelial cell.

28. A method of claim 26 wherein the selectin 5 mediates adhesion of a leukocyte to the endothelial cell.

29. A method of claim 26 wherein the selectin is ELAM-1. A method of claim 26 wherein the inflammatory

30. A method of cl disease process is psoriasis.

31. A method according to claim 1 for identifying a therapeutically effective blocking agent in a sample, substantially as hereinbefore described.

32. A blocking agent whenever identified by a method claimed in any one of claims 1-9 or'i31

33. A pharmaceutical composition according to claim