Inhibitors of MAdCAM-1-mediated interactions and methods of use therefor

Abstract

The present invention provides novel compounds comprising peptide sequences which mimic the conserved amino acid motif LDTSL of MAdCAM-1 and which have groups bonded to the N- and C-termini. Also provided are methods of inhibiting the interaction of a cell bearing a ligand of MAdCAM-1, such as human &agr;4&bgr;7, with MAdCAM-1 or a portion thereof (e.g., the extracellular domain), comprising contacting the cell with a compound of the present invention.

Description

RELATED APPLICATIONS

[0001] This application is a Continuation of U.S. Ser. No. 09/109,879 filed on Jul. 2, 1998, which is a Continuation of International Application No. PCT/US97/002911 filed on Jan. 3, 1997, which designates the United States, and which is a Continuation-In-Part of U.S. Ser. No. 08/582,740, filed Jan. 4, 1996, now U.S. Pat. No. 6,037,324, the teachings of all of which are incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

BACKGROUND OF THE INVENTION

[0003] Lymphocyte homing from the circulation to the lymphoid tissues and migration to sites of inflammation is regulated by interaction with receptors expressed in postcapillary venules, including high endothelial venules (HEV) found in secondary lymphoid tissues (e.g., mesenteric lymph nodes, Peyer's Patches (PP)) (Bevilacqua, M. P., Annu. Rev. Immunol., 11:767-804 (1993); Butcher, E. C., Cell, 67: 1033-1036 (1991); Picker, L. J., et al., Annu. Rev. Immunol., 10:561-591 (1992); and Springer, T. A., Cell, 76: 301-314 (1994)). These interactions are tissue specific in nature.

[0004] Inflammation (e.g., chronic inflammation) is characterized by infiltration of the affected tissue by leukocytes, such as lymphocytes, lymphoblasts, and mononuclear phagocytes. The remarkable selectivity by which leukocytes preferentially migrate to various tissues during both normal circulation and inflammation results from a series of adhesive and activating events involving multiple receptor-ligand interactions as proposed by Butcher and others (Butcher, E. C., Cell, 67: 1033-1036 (1991); vonAdrian, U. H. , et al., Proc. Natl. Acad. Sci. USA, 88:7538 (1991); Mayadas, T. N., et al., Cell, 74:541 (1993); (Springer, T. A. , Cell, 76:301 (1994)). As an initial step, there is a transient, rolling interaction between leukocytes and endothelium, which results from the interaction of selectins (and by &agr;4 integrins in some instances) with their carbohydrate ligands. This interaction, which is characterized by rolling in the direction of flow, can be assessed by known methods (Lawrence, M. B. and T. A. Springer, Cell, 65:859 (1991); WO 92/21746, Springer et al., (Dec. 10, 1992)). This is followed by activation events mediated by chemoattractants such as chemokines and their receptors, which cause activation of integrin adhesiveness and influence the direction of migration of leukocytes through vascular walls. Such secondary signals in turn trigger the firm adhesion of leukocytes to endothelium via leukocyte integrins and their endothelial ligands (Ig-like receptors and the ECM), and subsequent transendothelial migration from the circulation across the vascular endothelium.

[0005] In secondary lymphoid tissues, such as Peyer's patches (PPs) and lymph nodes (e.g., peripheral lymph nodes (PLN)), leukocyte trafficking and homing is regulated by interactions of homing receptors on the surface of leukocytes with endothelial cells lining the post-capillary venules, notably high endothelial venules (HEV) (Gowans, J. L. and E. J. Knight, Proc. R. Soc. Lond., 159:257 (1964)). Receptors termed vascular addressing, which are present on the endothelial cell surface and regulate the migration and subsequent extravasatlon of lymphocyte subsets. The vascular addressing show restricted patterns of expression and this tissue specific expression makes an important contribution to the specificity of leukocyte trafficking (Picker, L. J. and E. C. Butcher, Annu. Rev. Immunol., 10:561-591 (1992); Berg, E. L., et al., Cellular and molecular mechanisms of inflammation, 2:111 (1991); Butcher, E. C., Cell, 67:1033-1036 (1991)).

[0006] Mucosal vascular addressin MAdCAM-1 (Mucosal Addressin Cell Adhesion Molecule-1) is an immunoglobulin superfamily adhesion receptor for lymphocytes, which is distinct from VCAM-1 and ICAM-1. MAdCAM-1 was identified in the mouse as a −60 kd glycoprotein which is selectively expressed at sites of lymphocyte extravasation. In particular, MAdCAM-1 expression was reported in vascular endothelial cells of mucosal tissues, including gut-associated tissues or lymph of the small and large intestine, and the lactating mammary gland, but not in peripheral lymph nodes. MAdCAM-1 is involved in lymphocyte binding to Peyer's Patches. (Streeter, P. R., et al., Nature, 331:41-46 (1988); Nakache, M., et al., Nature, 337: 179-181 (1989); Picker, L. J., et al., Annu. Rev. Immunol., 10:561-591 (1992); Briskin, M. J., et al., Nature, 363:461 (1993); Berg, E. L., et al., Nature, 366:695-698 (1993); Berlin, C., et al., Cell, 74:185-195 (1993)). MAdCAM-1 can be induced in vitro by proinflammatory stimuli (Sikorski, E. E., et al., J. Imnunol., 151:5239-5250 (1993)). cDNA clones encoding murine and primate (e.g., human) MAdCAM-1 have been isolated and sequenced (Briskin, M. J. et al., Stature, 363: 461-464 (1993); Briskin et al., WO 96/24673, published Aug. 15, 1996; and Briskin, M. J. et al., U.S. Ser. No. 08/523,004, filed Sep. 1, 1995, the teachings of each of which is incorporated herein by reference in its entirety).

[0007] MAdCAM-1 specifically binds the lymphocyte integrin &agr;4&bgr;7 (also referred to as LPAM-1 (mouse), &agr;4&bgr;p (mouse)), which is a lymphocyte homing receptor involved in homing to Peyer's patches (Berlin, C., et al., Cell, 80:413-422 (1994); Berlin, C., et al., Cell, 74:185-195 (1993); and Erle, D. J., et al., J. Immunol., 153: 517-528 (1994)). In contrast to VCAM-1 and fibronectin, which interact with both &agr;4&bgr;1 and &agr;4&bgr;7 (Berlin, C., et al., Cell, 74: 185-195 (1993); Strauch, U. S., et al., Int. Immunol., 6:263 (1994)), MAdCAM-1 is a selective ligand for &agr;4&bgr;7 receptor.

[0008] Inflammatory bowel disease (IBD), such as ulcerative colitis and Crohn's disease, for example, can be a debilitating and progressive disease involving inflammation of the gastrointestinal tract. Affecting an estimated two million people in the United States alone, symptoms include abdominal pain, cramping, diarrhea and rectal bleeding. IED treatments have included anti-inflammatory drugs (such as corticosteroids and sulfasalazine), immunosuppressive drugs (such as 6-mercaptopurine, cyclosporine and azathioprine) and surgery (such as, colectomy). Podolsky, New Engl. J. Med., 325:928-937 (1991) and Podolsky, New Engl. J. Med., 325:1008-1016 (1991). There is a need for inhibitors of MAdCAM-1 function to provide new therapies useful in the treatment of IED and other diseases involving leukocyte infiltration of the gastrointestinal tract or other mucosal tissues.

SUMMARY OF THE INVENTION

[0009] As shown herein, the conserved amino acid motif LDTSL (SEQ ID NO:1) is involved in Mucosal Addressin Cell Adhesion Molecule-1 (hereinafter “MAdCAM-1”) binding to MAdCAM-1 ligands, such as human &agr;4&bgr;7. In addition, compounds containing this peptide sequence or truncated versions thereof, e.g., Asp-Thr and Leu-Asp-Thr, bind to &agr;4&bgr;7 and can inhibit adhesion of leukocytes expressing &agr;4&bgr;7 on the cell surface to MAdCAM-1. It has also been discovered that groups on the N- and C-termini of the peptide sequences enhance the binding of these compounds to &agr;4&bgr;7 and are more potent inhibitors of interaction between MAdCAM-1 and its ligands. Accordingly, the present invention provides novel compounds comprising peptide sequences which mimic the conserved amino acid motif LDTSL and which have groups bonded to the N- and C-termini.

[0010] Also provided are methods of inhibiting the interaction of a cell bearing a ligand of MAdCAM-1, including &agr;4&bgr;7 integrins, with MAdCAM-1 or a portion thereof (e.g., the extracellular domain), comprising contacting the cell with a compound of the present invention. In one embodiment, the invention relates to a method of inhibiting the MAdCAM-mediated interaction of a first cell bearing an &agr;4&bgr;7 integrin with MAdCAM, for example with a second cell bearing MAdCAM, comprising contacting the first cell with a compound of the present inventLon. In another embodiment, the invention relates to a method of treating an individual suffering from a disease associated with leukocyte recruitment to tissues (e.g., endothelium) expressing the molecule MAdCAM-1.

[0011] One embodiment of the present invention is a method of inhibiting the binding of a cell such as a leukocyte expressing a ligand for MAdCAM-1 on the cell surface (e.g., &agr;4&bgr;7) to MAdCAM-1, for example to endothelial cells expressing MAdCAM-1 on the cell surface. The method comprises contacting the leukocytes with an effective amount of an inhibitor represented by Structural Formula (I):

R1—X—Y—Z—R2  (I)

[0012] Y is a pentapeptide [AA]1-[AA]2-[AA]3-[AA]4—[AA]5.

[0013] [AA]1 is selected from the group consisting of leucine, valine, isoleucine, alanine, phenylalanine, glycine, N-methylleucine, serine, threonine, ornithine, cysteine, aspartic acid, glutamic acid and lysine.

[0014] [AA]2 is selected from the group consisting of aspartic acid, glutamic acid, phenylalanine and tyrosine.

[0015] [AA]3 is selected from the group consisting of threonine, serine, valine, proline and 4-hydroxyproline.

[0016] [AA]4 is selected from the group consisting of serine, cysteine, aspartic acid, glutamic acid, proline, 4-hydroxyproline, threonine, valine, isoleucine, alanlne, glycine, ornithine and lysine.

[0017] [AA]5 is selected from the group consisting of leucine, valine, isoleucine, N-methylleucine, threonine, ornithine, serine, alanine, glycine, phenylalanine, cysteine, aspartic acid, glutamic acid and lysine.

[0018] X and Z are independently chosen from the group consisting of a covalent bond, an amino acid or a peptide. Each amino acid in X and Z is independently selected from the group of naturally occurring amino acids. X and Z are preferably covalent bonds.

[0019] R1 is R3—CO—.

[0020] R2 is —NR4R .

[0021] R3 is selected from the group consisting of a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group.

[0022] R4 and R5 are each independently selected from the group consisting of hydrogen, a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group. R4 and R5 are not both —H. Taken together, R4 and R5 can also form a heterocyclic ring.

[0023] Taken together, X, Y and Z form a peptide containing no more than about fifteen amino acid residues.

[0024] The compound represented by Structural Formula (I) is a peptide having a group bonded to the nitrogen atom at the N-terminus and a second group bonded to the carbonyl at the C-terminus. In one aspect, the peptide is linear. The N-terminus of Y is not bonded to arginine or an arginine derivative.

[0025] Optionally, the peptide formed by X, Y and Z is cyclized to form a ring. When cyclized, the ring can be formed by an amide linkage, an ester linkage or a dlsulfide linkage between two amino acids in the peptide. When the ring is formed by an amide linkage between the N-terminus and C-terminus of the peptide, R1 and R2 together form a covalent bond between the carbonyl at the C-terminus of the peptide and the nitrogen at the N-terminus of the peptide.

[0026] In another embodiment of the method of inhibiting the binding of a cell such as a leukocyte expressing a ligand for MAdCAM-1 on the cell surface (e.g., &agr;4&bgr;7) to MAdCAM-1, for example endothelium expressing the molecule MAdCAM-1, the inhibitor administered is represented by Structural Formula (II):

R1—X—Y′—Z—R2  (II)

[0027] R1, R2, X and Z are as defined for Structural Formula (I). Y′ represents a dipeptide having the sequence Asp-Thr, a tripeptide having the sequence Leu-Asp-Thr, or a pentapeptide [AA]1-[AA]2-[AA]3-[AA]4-[AA]5 having the sequence Leu-Asp-Thr-Ser-Leu (SEQ ID NO:1) with the proviso that any single one of [AA]1, [AA]2, [AA]3, [AA]4 or [AA]5 can vary, being any naturally occurring amino acid. For example, the pentapeptide can be Xaa-Asp-Thr-Ser-Leu (SEQ ID NO.: 85), Leu-Xaa-Thr-Ser-Leu (SEQ ID NO: 86), Leu-Asp-Xaa-Ser-Leu (SEQ ID NO: 87), Leu-Asp-Thr-Xaa-Leu (SEQ ID NO: 88), or Leu-Asp-Thr-Ser-Xaa (SEQ ID NO: 89). The nitrogen at the N-terminus of Y′ may be bonded to any naturally occurring amino acid (including arginine) with the proviso that the N-terminus of Y′ may not be bonded to glycine or sarcosine when Y′ is Asp-Thr and the peptide formed from X—Y′—Z is cyclized, as described below.

[0028] X, Y′ and Z taken together form a peptide containing no more than about fifteen amino acids. In one aspect, the peptide formed by X, Y′ and Z is linear. Preferably X and Z are each a covalent bond. Optionally, the peptide formed by X, Y′ and Z is cyclized to form a ring. When cycllzed, the ring can be formed by an amide linkage, an ester linkage or a disulfide linkage between two amino acids in the peptide. When the ring is formed by an amide linkage between the N-terminus and C-terminus of the peptide, R1 and R2 together form a covalent bond between the carbonyl at the C-terminus of the peptide and the nitrogen at the N-terminus of the peptide.

[0029] Another embodiment of the present invention is a novel compound. The compound is represented by Structural Formula II, as described above.

[0030] Another embodiment, of the present invention is a method of treating an individual suffering from a disease associated with leukocyte infiltration of tissues expressing the molecule MAdCAM-1. The method comprises administering to the individual a therapeutically effective amount of an inhibitor represented by Structural Formula (I) or an inhibitor represented by Structural Formula (II).

[0031] Compounds of the present invention are inhibitors of the binding of MAdCAM-1 to the receptor &agr;4&bgr;7 and are therefore useful in the treatment of diseases such as inflammatory bowel disease with the potential for fewer side effects in other tissues where adhesion is mediated by &agr;4&bgr;1 integrin, for example.

[0032] The compounds of the present invention are also useful in diagnostic and research applications. For example, the compounds can be used as immunogens (e.g., when conjugated to a suitable carrier) to induce the formation of antibodies which selectively bind MAdCAM-1 or a portion thereof. These antibodies can in turn be used to identify cells expressing MAdCAM-1 on their cell surface or detect MAdCAM-1 in a sample. In addition, the compounds of the present invention can be labelled and used to detect &agr;4&bgr;7 integrin and/or quantitate expression of &agr;4&bgr;7 integrin on the surface of cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 is a graph illustrating a titration experiment used to select the amount of rat anti-murine k capture antibody which was used in adhesion assays. MAdCAM-mCk fusion protein was bound to the surface of a well via various amounts of capture antibody, and the adhesion of fluorescently labelled HUT 78 cells to fusion protein in the presence of increasing amounts of anti-MAdCAM-1 antibody was monitored. Reading (Y-axis) is in arbitrary fluorescence units. 50 &mgr;l of a 10 &mgr;g/ml (▪), 5 &mgr;g/ml (□), 2.5 &mgr;g/ml (♦) or 1.25 &mgr;g/ml (⋄) solution of purified rat anti-murine k antibody was used. Anti-murine MAdCAM-1 antibody MECA-367 was used as neat supernatant or diluted 1:2, 1:4, 1:8 or 1:16 (indicated on the X-axis by :1 (neat), :2, :4, :8, and :16, respectively). “None” indicates no MECA-367 antibody (buffer control) was present.

[0034] FIG. 2 is a graph illustrating the inhibition by compound 793-la of the adherence of fluorescently labelled cells bearing &agr;4&bgr;7 (HUT 78 human T cell lymphoma cells) to a MAdCAM-mCk fusion protein bound by capture antibody. The various concentrations (&mgr;M) of compound used were plotted against a ratio (calculated from measurements of adherent fluorescent cells; see Example 2).

[0035] FIG. 3 is a schematic illustration of a MAdCAM-1/mCk fusion gene and encoded fusion protein. A BamHI site at the junction of the MAdCAM-1 and mCk sequences introduces codons for Gly-Ser (SEQ ID NO: 63 and SEQ ID NO: 64). The fusion gene encodes the signal peptide (SEQ ID NO: 65 and SEQ ID NO: 66, partial signal peptide) and complete extracellular domain of MAdCAM-1 through the threonine residue at posItion 365. Single letter amino acid codes are used.

[0036] FIG. 4 is an illustration of the nucleotide sequence determined from subclones of cDNA clone 4 encoding human MAdCAM-1 (SEQ ID NO: 67), and the sequence of the predicted protein encoded by the open reading frame (MAdCAM-1) (SEQ ID NO: 68). The predicted signal peptide and transmembrane region are underlined in bold. Cysteine residues of the two Ig-like domains are boxed, as are potential N-linked glycosylation sites. The mucin domain consisting of 71 amino acids is outlined by a thin bold line. The LDTSL (SEQ ID NO: 1) motif begins at amino acid 63.

[0037] FIG. 5 is an illustration of the nucleotide sequence determined from subclones of cDNA clone 20 encoding human MAdCAM-1 (SEQ ID NO: 69), and the sequence of the predicted protein encoded by the open reading frame (MAdCAM-1) (SEQ ID NO: 70). The predicted signal peptide and transmembrane region are underlined in bold. Cysteine residues of the two Ig-like domains are boxed, as are potential N-linked glycosylation sites. The mucin domain consisting of 47 amino acids is outlined by a thin bold line. The LDTSL (SEQ ID NO: 1) motif begins at amino acid 63. The two human cDNA clones are probably isoforms encoded by genomic DNA, generated, for example, by alternative splicing or by transcription of two different alleles.

DETAILED DESCRIPTION OF THE INVENTION

[0038] As used herein, “lower alkyl” refers to a hydrocarbon containing from one to about twelve carbon atoms. In one aspect, the term “C3-C12 alkyl” is employed to include all lower alkyls except —CH3 and —CH2—CH3. The hydrocarbon can be saturated or can contain one or more units of unsaturation. The hydrocarbon can also be branched, straight chained or cyclic.

[0039] A “cyclic hydrocarbon” refers to a cycloalkyl group. A “cycloalkyl group” includes carbocyclic rings (e.g., cyclopentyl and cyclohexyl) as well as carbocyclic rings in which one or more carbon atoms is replaced by a heteroatom (e.g., morpholino, piperidinyl, pyrrolidinyl, thiomorpholino or piperazinyl), Also included are cycloalkyl rings fused to other cycloalkyl rings (e.g., decalinyl) or fused to aromatic or heteroaromatic groups (e.g., indanyl, tetralinyl and 9-xanthenyl). A “cyclic hydrocarbon” also includes bridged polycyclic structures such as adamantyl and nonbornyl. As with other lower alkyl groups, a cyclic hydrocarbon can be substituted.

[0040] As used herein, “aryl” groups are defined to include aromatic ring systems such as phenyl. “Heteroaryl” groups are defined to include aromatic ring systems which contain heteroatoms, such as 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 2-pyridyl, 2-pyranyl and 3-pyranyl. “Aryl” and “heteroaryl” groups also include fused polycyclic aromatic ring systems in which the aryl or heteroaryl ring is fused to one or more other aryl, heteroaryl or cycloalkyl rings. Examples include a-naphthyl, &bgr;-naphthyl, 1-anthracenyl, 2-anthracenyl, 2-benzothienyl, 3-benzothienyl, 2-benzofuranyl, 3-benzofuranyl, 2-indolyl, 2-quinolinyl, 3-quinolinyl, acridinyl and 9-thioxanthanyl. Also included are polycyclic aromatic ring systems in which aryl groups, heteroaryl groups or an aryl and a heteroaryl group are connected by a covalent bond (e.g., 4-phenylphenyl, 3,5-diphenylphenyl, 4-(2-thienyl)phenyl and 4-(2-furanyl)phenyl)) or a —(CH)n— bridge (e.g., 4-(phenyl-CH2)phenyl, 4-(phenylCH2CH2-) phenyl, 4-(-CH2-2-thienyl)phenyl and 4-(-CH2CH2-2-thienyl)phenyl), wherein n is an integer from 1 to about 5.

[0041] Suitable subscituents on a lower alkyl group, an aryl group or a heteroaryl group include C1-C2 alkoxy, ketone, aldehyde, (lower alkyl) ester, aryl, substituted aryl, heteroaryl, substituted heteroaryl benzyl, lower alkyl, fluoro, bromo, iodo, cyano, nitro and the like. A lower alkyl group, an aryl group or a heteroaryl group may have more than one substituent (e.g., 2,2,3,3-tetramethylcyclopropyl, 3,5-diphenylphenyl and 2,4-dichlorophenyl), 2-bromo-4-nitro-pentyl and 2-(3,5-dibromo-benzofuranyl). In addition, a substituted lower alkyl group can have multiple substituents on one carbon atom (e.g., diphenylmethyl, triphenylmethyl, 2,2,3,3-tetramethylcyclopropyl and trifluoromethyl).

[0042] R1 and R2 are groups covalently bonded to the N-terminal and the C-terminal, respectively, of the peptide sequences of the compounds represented by Structural Formulas (I) and (II).

[0043] In one preferred embodiment, R3 is selected from the group consisting of adamantyl, adamantylmethyl, a C1-C4 alkyl group, a C3-C7 cycloalkyl group, a C3-C7 cycloalkyl group substituted with a C1-C4 alkyl group, an aryl group substituted with a C3-C7 cycloalkyl group, phenyl substituted with a C1-C8 alkyl group, an aryl group, 2-anthracenyl, diphenylmethyl, 1-napthyl, 2-naphthyl, benzyl, indanyl, tetralinyl, triphenylmethyl, triphenyl (C1-C4 alkyl), 9-fluorenyl, styryl, a heteroaryl group, furanyl, thienyl, 9-xanthanyl, 9-thioxanthanyl, acridinyl, pyridyl, quinolinyl, a C1-C4 alkyl group substituted with an aryl group (e.g.,, benzyl and phenylethyl) and a C1-C4 alkyl group substituted with heteroaryl (furanylmethyl, thienylmethyl, pyridylmethyl, quinolinylmethyl). More preferably, R3 is selected from the group consisting of triphenylmethyl, diphenylmethyl, 3,5-diphenylphenyl, 2-furanyl, 3-furanyl, 9-xanthenemethyl, 2,2,2-triphenylethyl, 2-anthracene, methyl, cyclopentyl, 2-indolyl, 2-indanyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, cyclohexyl, 5-phenylpentyl, 4-isobutyl-&agr;-methylphenylmethyl, 4-biphenylmethyl, &agr;-naphthylmethyl, 4-heptylphenyl, phenylmethyl, trans 2-phenylethenyl and 2,2,3,3-tetramethylcyclopropyl.

[0044] In another preferred embodiment, R3 is a substituted or unsubstituted aryl or heteroaryl group. Examples include monocyclic and bicyclic nitrogen-containing heteroaromatic groups, such as a quinolinyl group (e.g., 2-quinolinyl, 2-phenyl-4-quinolinyl, 3-quinolinyl, 4-quinolinyl, 7-quinolinyl), an isoquinolinyl group (e.g., 3-isoquinolinyl), an indolyl group (e.g., 2-indolyl and 5-chloro-2-indolyl), a quinoxalinyl group (e.g., 2-quinoxalinyl), a cinnolinyl group, and a pyrazinyl group. Also included are vinyl groups substituted with substituted or unsubstituted aryl or heteroaryl groups (e.g., cis and trans styryl and stilbyl, (3-pyridyl) —CH═CH—), polycarbocyclic aromatic hydrocarbons (e.g., 2-naphthyl and 2-anthracyl) and oxygen-containing polycyclic aromatic hydrocarbons (e.g., 9-xanthanyl, 2-benzopyranone, 3-benzopyranone and 2-benzofuranyl). Suitable substituents for an aryl or heteroaryl group are as described above.

[0045] R4 and R5 are preferably independently selected from the group consisting of a C1-C5 alkyl group, a C1-C5 alkyl group substituted with C1-C4 alkoxy, a C3-C7 cycloalkyl group, a C3-C7 cycloalkyl group substituted with a C1-C5 alkyl group, an aryl group, a substituted C1-C5 alkyl group (e.g.,, suitable substituents include a hydroxyl group, phenyl, substituted phenyl (e.g., suitable substituents include a C1-C5 alkyl group, C1-C4 alkoxy, halogen, nitro and trifluoromethyl)), a C1-C4 alkyl group substituted with an aryl group (e.g., benzyl, phenylethyl, diphenylethyl, 9-fluorenyl), a heteroaryl group (e.g., benzofuranyl, benzothienyl, furanyl, pyridiryl, auinolinyl, and thienyl) and a C1-C5 alkyl group substituted with a heteroaryl group (e.g., furanylmethyl, thienylmethyl, pyridylmethyl and quinolinylmethyl).

[0046] In addition, R4 and R5 may, taken together, form a heterocyclic ring including piperidinyl, pyrrolidinyl, morpholino, thiomorpholino or piperazlnyl where the N4-position of the piperazine ring is substituted by a group consisting of hydrogen, acetyl, benzoyl, alkyl, benzyl or phenyl. More preferably, R4 and R5 are eacn independently selected from the group consisting of 2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, —CH2-2-thienyl, —CH2-3-thienyl, —CH2-2-furanyl, —CH2-3-furanyl, 3,4-dimethoxybenzyl, and isopentyl.

[0047] Examples of compounds of the present invention include the following:

[0048] Cpc-NH-Asp-Thr-N(CH2CH2OH) (Benzyl)

[0049] Cpc-NH-Asp-Thr-NH-Hexyl

[0050] Cpc-NH-Asp-Thr-NH-(3,4-Dimethoxybenzyl)

[0051] Cpc-NH-Asp-Thr-NH-CH(Phenyl)2

[0052] Idc-NH-Asp-Thr-NH-Benzyl

[0053] Chc-NH-Asp-Thr-NH-CH2Thienyl

[0054] Bpc-NH-Asp-Thr-NH-Benzyl

[0055] Indc-NH-Asp-Thr-NH-Isopentyl

[0056] Bpc-NH-Asp-Thr-NH-CH2Thienyl

[0057] Abbreviations are defined in Table 3.

[0058] In a preferred embodiment, R3 is selected from the group consisting of diphenylmethyl, triphenylmethyl, trans 2-phenyl-ethylenyl, 2-phenyl-ethynyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl and 3-benzothienyl, R4 is selected from the group consisting of 2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, —CH2-2-thienyl, —CH2-3-thienyl, —CH2-2-furanyl, —CH2-3-furanyl and P, is —H.

[0059] As used herein, “[AA]” represents an amino acid and “[AA]1. . . [AA]n” represents a peptide, wherein [AA]1 is the N-terminal amino acid and [AA]n is the C-terminal amino acid. Unless otherwise indicated, amino acids in the peptides are presented from left to right, with the left end being the N-terminus and the right end being the C-terminus. The amino acids within the peptide can be similarly designated. For example, “[AA]2” and “[AA]n-1” refer to the second amino acid from the N-terminal and C-terminal, respectively. This nomenclature permits each amino acid of X and Z to be designated. For example,, “[AA]1 of Z” refers to the amino acid at the N-terminus of Z when Z is a peptide. “[AA]2 of X” refers to the second amino acid from the N-terminus of X when X is a peptide. “[AA]n-1 of X” refers to the second amino acid from the C-terminus of X when X is a peptide. It is to be understood that the amino acids in Y are numbered in sequence from 1-5 (Y′ is numbered in sequence from 1-5, 1-3 or 1-2 when Y′ is a pentapeptide, a tripeptide or a dipeptide, respectively) beginning at the N-terminus and ending at the C-terminus. Thus, “[AA]2 of Y” refers to the second amino acid from the N-terminal of Y. It is also to be understood that “[AA]1 of X” (or “[Aa]1 of Z”) can be used to represent X (or Z) when X (or Z) is a single amino acid.

[0060] In one embodiment Y is Y′, e.g., Asp-Thr, Leu-Asp-Thr or a pentapeptide [Aa1-[AA]2-[AA]3-[AA]4-[AA]5 having the sequence Leu-Asp-Thr-Ser-Leu (SEQ ID NO: 1) with the proviso that a single one of [AA]1, [AA]2, [AA]3, [AA]4 or [AA]5 can vary, being any naturally occurring amino acid. For example, the pentapeptide can be Xaa-Asp-Thr-Ser-Leu (SEQ ID NO.: 85), Leu-Xaa-Thr-Ser-Leu (SEQ ID NO: 86), Leu-Asp-Xaa-Ser-Leu (SEQ ID NO: 87), Leu-Asp-Thr-Xaa-Leu (SEQ ID NO: 88), or Leu-Asp-Thr-Ser-Xaa (SEQ ID NO: 89). A “naturally occurring amino acid” is an amino acid that occurs in nature. “Naturally occurring amino acids” includes, but are not limited to serine, threonine, cysteine, glycine; valine, alanine, leucine, isoleucine, aspartic acid, glutamic acid, glutamine, asparagine, lvsine, arginine, ornithine, tyrosine, phenylalanine, histidine, proline, 4-hydroxyproline, tryptophan and methionine.

[0061] For Structural Formula (I), if X is an amino acid, X may not be arginine or a derivative of arginine. In addition, for Structural Formula (I) if X is a peptide, the amino acid at the C-terminus of X may not be arginine or an arginine derivative. Arginine derivatives include N and/or N′ C1-C4 alkylated arginines (e.g., N-alkyl arginine, N,N-dialkyl arginine, N,N′-dialkyl arginine, and N,N,N′-trialkyl arginine). Arginine derivatives also include arginine mimics (e.g., p-aminomethyl arginine), arginine isomers (e.g., norarginine), arginine having substituents in the side chain (e.g., nitro, halo or C1-C4 alkyl) and arginines containing one or more additional carbon atoms in the side chain (e.g., homoarginine).

[0062] In a preferred embodiment, Y′ is the tripeptide Leu-Asp-Thr or the dipeptide Asp-Thr, and examples of preferred groups for R3 include substituted and unsubstituted phenyl (e.g., −2,5-diCF3, 2-CF3, 3-CF3, 1,2-difluoro, 3,5-difluoro, 2,5,6-trifluoro, 3-(n-hexyloxy), 3-chloro, 4-t-butyl and 4-phenyl substituted phenyl), thienyl (e.g., 2-thienyl and 3-halo-2-thienyl), indolyl (e.g., 2-indolyl), pyrimidyl (e.g., 1-chlor-3-trifluoro-4-pyrimidyl), pyridyl (e.g., 2-methyl-5-chloro-3-pyridyl), benzofuranyl (e.g., 2-benzofuranyl), isoqzuinlinyl (e.g., 3-isoquinolinyl), 3-isoquinolinyl-CO—NH—(CH2)x— (wherein x is an integer from 1-4) and benzopyranone groups (e.g., 2- and 3-benzopyranone). R3 is more preferably 3-isoquinolinyl or 2-benzoluranyl. R4 is preferably —H and R5 is preferably benzyl; substituted benzyl (e.g., 3-CF3, 3-methyl, 4-CH3, 2-CH3, 4-fluoro, 3,4-difluoro, 2,5-difluoro, 3,4-methylenedioxy, 4-N-morpholino and 4-OCH3 substituted benzyl), phenethyl, substituted phenethyl (e.g., 3,4-dimethoxy and 4-SO2NH2 substituted phenethyl), phenpropyl, substituted phenpropyl, heteroaryl-CH2— (wherein heteroaryl is, e.g., 3-furanyl, 3-thienyl and 4-pyridinyl), substituted heteroaryl-CH2— (e.g., 6-(2-methyl)-quinolinyl), lower alkyl, substituted lower alkyl (e.g., isopropyl, methoxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 3-(2-methyl-N-piperidinyl)propyl, 1-(ethyl)-1-propyl, 2-(1-cyclohexene)ethyl and 1-(cyclohexyl)eethyl) and cycloalkyl (e.g., cyclopropyl, cyclopentyl, cyclohexyl and cyclooctyl). Other examples of R5 are represented by the following structural formulas: 1

[0063] In addition, taken together, R4 and R5 can form a heterocyclic ring such as pyrrolidinyl, substituted pyrrolidinyl (e.g., 3,3-dimethyl substituted and 3-dimethylamino substituted pyrrolidinyl), indoline, isomers of indoline, substituted indoline, substituted isomers of indoline, tetrahydroisoquinoline, substituted tetrahydroisoquinoline, tetrahydroquinoline, substituted tetrahydroquinoline, piperidones (e.g., 4-piperidone), substituted piperidones, piperidine, substituted piperidines, tetrahydro-oxazines (e.g., 1,3-tetrahydro-oxazine and morpholine) and substituted tetrahydro-oxazines (e.g., 2,4-dimethyl-1,3-tetrahydro-oxazine and 3,5-dimethyl morpholine).

[0064] In another embodiment, when Y′ is the tripeptide Leu-Asp-Thr or the dipeptide Asp-Thr, R1 is represented by Structural Formula (IV): 2

[0065] A is an aryl group, a substituted aryl group a heteroaryl group or a substituted heteroaryl group, as described above. Examples include 3-isoquinolinyl, 2-indolyl, 5-chloro-2-indolyl, 2-benzofuranyl, phenyl and trans-styryl. n and m are each independently zero or one. Preferably, n+m=1.

[0066] The substitution pattern on the phenyl ring in Structural Formula (IV) can be ortho, meta or para.

[0067] In another preferred embodiment Y or Y′ is the pentapeptide [AA]-[AA]2-[AA]3-[AA]4-[AA]5] wherein:

[0068] amino acid [AA]1 is selected from the group consisting of leucine, isoleucine, alanine, valine, glycine, phenylalanine and N-methylleucine;

[0069] amino acid [AA]2 is selected from the group consisting of aspartic acid, glutamic acid, phenylalanine and tyrosine;

[0070] amino acid [AA]3 is selected from the group consisting of threonine, serine, valine, proline and 4-hydroxyoroline;

[0071] amino acid [AA]4 is selected from the group consisting of serine, cysteine and threonine; and

[0072] amino acid [AA]5 is selected from the group consisting of leucine, alanine, valine, isoleucine, alanine, glycine, phenylalanine and N-methylleucine.

[0073] In another aspect, any one or more of [AA]1, [AA]2, [AA]3, [AA]4 and [AA]5 in the pentapeptide of Y or Y′ can also be a non-naturally occurring amino acid. [AA]1 and/or [AA]5 can be a non-naturally occurring amino acid in which the side chain is a C2-C7 substituted or unsubstituted alkyl group or a substituted phenyl group. Side chain alkyl groups can be straight chained, branched or cyclic. Suitable substituents for an alkyl side chain include non-polar groups such as C1-C2 alkyl, halo, cyano, C1-C3 alkoxy, phenyl or substituted phenyl. Suitable substituents for a side chain phenyl group include non-polar groups such as C1-C2 alkyl, halo, cyano or —C1-C3 alkoxy. [AA]2 can be a non-naturally occurring amino acid having a C3-C6 straight chained or branched alkyl group substituted with a carboxylic acid. [AA]3 and/or [AA]4 can be a non-naturally occurring amino acid in which the side chain is a C2-C6 straight chained or branched alkyl group substituted with an alcohol or thiol.

[0074] The compounds represented by Structural Formulas (I) and (II) are peptides X-Y-Z or X-Y-Z, respectively, with groups attached to the N-terminus and C-terminus. Preferably, X and Z are covalent bonds, i.e. the compounds represented by Structural Formulas (I) and (II) are peptides Y or Y′, respectively, with group R attached to the N-terminal of Y or Y′, and with group R2 attached to the C-terminal of Y or Y′. In one aspect, X-Y-Z or X-Y′-Z is a linear peptide. In another aspect, X-Y-Z or X-Y′-Z is cyclized.

[0075] As used herein, “cyclized” refers to forming a ring by a covalent bond between suitable side chains of two amino acids in the peptide. For example, a disulfide can be formed between the sulfur atoms in the side chains of two cysteines. Alternatively, an ester can be formed between the carbonyl carbon in the side chain of, for example, glutamic acid or aspartic acid, and the oxygen atom in the side chain of, for example, serine or threonine. An amide can be formed between the carbonyl carbon in the side chain of, for example, glutamic acid or aspartic acid, and the amino nitrogen in side chain of, for example, lysine or ornithine.

[0076] “Cyclized” also refers to forming a ring by a peptide bond between the nitrogen atom at the N-terminus and the carbonyl carbon at the C-terminus. In this case, R1 and R2 together form a covalent bond.

[0077] “Cyclized” also refers to forming a ring by forming a covalent bond between the nitrogen at the N-terminus of the compound and the side chain of a suitable amino acid in the peptide. For example, an amide can be formed between the nitrogen atom at the N-terminus and the carbonyl carbon in the side chain of aspartic acid or glutamic acid. Alternatively, the compounds represented by Structural Formulas (I) and (II) can also be cyclized by forming a covalent bond between the carbonyl at the C-terminus of the compound and the side chain of a suitable amino acid in the peptide. For example, an amide can be formed between the carbonyl carbon at the C-terminus and the amino nitrogen atom in the side chain of lysine or ornithine; an ester can be formed between the carbonyl carbon at the C-terminus and the hydroxyl oxygen atom in the side chain of serine or threonine.

[0078] Preferably, the ring of the cyclized compounds of the present invention contains four to nine amino acids. The peptide is preferably cyclized to maximize within the ring the number of amino acids involved in the binding of the peptide to a ligand of MAdCAM-1, such as human &agr;4&bgr;7. Thus, the ring contains at least four of the five amino acids in Y or Y′, when Y or Y′ is a pentapeptide. More preferably, the ring contains all of the amino acids in Y or Y′.

[0079] For compounds represented by Structural Formula (I), the ring can be formed by a bond between the side chain of an amino acid selected from the group consisting of [AA]n-3 of X, [AA]n-2 of X, [AA]n-1 of X, [AA]n of X (if the specific amino acid is present in X) and [AA]1 of Y and the side chain of any one of the amino acids selected from the group consisting of [AA]5 of Y, [AA]1 of Z, [AA]2 of Z, [AA]3 of Z and [AA]4 of Z (if the specific amino acid is present in (X and/or Z), with the proviso that the ring contains from four to nine amino acids. The ring can also be formed by a bond between the side chain of [AA]4 and the side chains of any one of [AA]n-4 of X, [AA]n-3 of X, [Aa]n-2 of X, [AA]n-1 of X, [AA]n of X (if the specific amino acid is present in X) or [AA]1 of Y when the ring contains only four of the five amino acids of Y. Alternatively, the ring can also be formed by a bond between the side chain of [AA]2 and the side chains of any one of [AA]5 of Y, [AA]1 of Z, [AA]2 of Z. [AA]3 of Z. [AA]4 of Z or [AA]5 of Z (if the specific amino acid is present in Z) when the ring contains only four of the five amino acids of Y.

[0080] For compounds represented by Structural Formula (II), when Y′ is a pentapeptide, the ring can be formed as described in the preceding paragraph. When Y′ is a tripeptide, the ring can be formed by a bond between the side chain functional group of an amino acid selected from the group consisting of [AA]n-5 of X, [AA]n-4 of X, [AA]n-3 of X, [AA]n-2 of X, [AA]n-1 of X, [AA]n of X and [AA]1 of Y′ and the side chain functional group of any one of the amino acids selected from the group consisting of [AA]3 of Y′, [AA]1 of Z, [AA]2 of Z, [AA]3 of Z, [AA]4 of Z, [AA]5 of Z and [AA]6 of Z (if the specific amino acid is present in X and/or Z), with the proviso that the ring contains from four to nine amino acids. When Y′ is a dipeptide, the ring can be formed by a bond between the side chain of an amino acid selected from the group consisting of [AA]2 of Y′, [AA]n-5 of X, [AA]n-4 of X, [AA]n-3 of X, [AA]n-2 of X, [AA]n-1 of X, [AA]n of X and [AA]1 of Y′ and the side chain functional group of any one of the amino acids selected from the group consisting of [AA]2 of Y′, [AA]1 of Z, [AA]2 of Z, [AA]3 of Z, [AA]4 of Z, [AA]5 of Z, [AA]6 of Z and [AA]7 of Z (if the specific amino acid is present in X and/or Z), with the proviso that the ring contains from four to nine amino acids.

[0081] When the ring contains all of the amino acids of Y or Y′, the compound of Structural Formula I or II can be cyclized by a peptide bond between the nitrogen at the N-terminus of X and the carbonyl carbon at the C-terminal of Z.

[0082] In one embodiment, X is an amino acid or has an amino acid sequence which is the same as that immediately N-terminal to the LDTSL (SEQ ID NO: 1) motif of human MAdCAM-1, and Z is an amino acid or has an amino acid sequence which is the same as that immediately C-terminal to the LDTSL (SEQ ID NO: 1) motif of human MAdCAM-1. In a further aspect, any one or two amino acids in X and Z can replaced by a naturally occurring amino acid or a non-naturally occurring amino acid, as described above.

[0083] Peptide sequences in the compounds of the present invention may be synthesized by solid phase peptide synthesis (e.g., BOC or FMOC) method, by solution phase synthesis, or by other suitable technlaues including combinations of the foregoing methods. The BOC and FMOC methods, which are established and widely used, are described in Merrifield, J. Am. Chem. Soc. 88:2149 (1963) Meienhofer, Horrmonal Proteins and Peptides, C. H. Li, Ed., Academic Press, 1983, pp. 48-267; and Barany and Merrifield, in The Peptides, E. Gross and J. Meienhofer, Eds., Academic Press, New York, 1980, pp. 3-285. Methods of solid phase peptide synthesis are described in Merrifield, R. B., Science, 232: 341 (1986); Carpino, L. A. and Han, G. Y., J. Org. Chem., 37: 3404 (1972); and Gauspohl, H. et al., Synthesis, 5: 315 (1992)). The teachings of these six articles are incorporated herein by reference in their entirety. Examples of the synthesis of the compounds having the structure represented by Structural Formulas (I) and (II) are disclosed in Examples 1, General Procedures A-C.

[0084] N- and C-terminal modified peptides can be prepared on an oxime resin using a modification of procedures described in DeGrado and Kaiser, J. Org. Chem. 47:3258 (1982), the teachings of which are incorporated herein by reference. Oxime resin can be obtained from Nova Biochem, Inc. The resin is acylated with, for example, Fmoc-Thr(t-Bu)-OH (4-6 equivalents), HBTU (4-6 equivalents)/NMM (8-12 equivalents) in dimethylformamide (DMF) at room temperature over about twenty hours. The resin is then washed with DMF, followed by methylene chloride and dried under vacuum prior to use. A peptide can then be synthesized using the Fmoc-Thr(t-Bu)-Oxime resin and General Procedure C in Example 1. The resulting peptide is reacted with the desired amine (8-12 equivalents) in methylene chloride for twelve hours. The resin is then washed with methylene chloride The filtrate is washed with 5% citric acid in water and dried over anhydrous magnesium sulfate. The solvent is removed and the resulting residue dried under vacuum to give the desired N- and C-terminal modified residue.

[0085] Methods of cyclizing compounds having peptide sequences are described, for example, in Lobl et al., WO 92/00995, the teachings of which are incorporated herein by reference in their entirety. Cyclized compounds can be prepared by protecting the side chains of the two amino acids to be used in the ring closure with groups that can be selectiveyv removed while all other side-chain protecting groups remain intact. Selective deprotection is best achieved by using orthogonal side-chain protection such as allyl (OAI) (for the carboxyl group in the side chain of glutamic acid or aspartic acid, for example), allyloxy carbonyl (Aloc) (for the amino nitrogen in the side chain of lysine or ornithine, for example) or acetamidomethyl (Acm) (for the sulfhydryl of cysteine) protecting groups. OAI and Ac are easily removed by Pd° and Acm is easily removed by iodine treatment. Examples of cyclizing a compound represented by Structural Formulas (I) and (II) by forming a disulfide bond are given in Example 1 and General Procedure D.

[0086] In another example, the cyclic peptide K*LDTSLD* (SEQ ID NO: 2), cyclized between the side chains of lysine and the N-terminal aspartic acid (“*” indicates a cyclizing amino acid) peptide can assembled on PAL-PEG-PS-resin using Nu-Fmoc-amino acids and t-butyl side-chain protection as described in General Procedures A and B in Example 1, except D* and K* are incorporated in the linear chain as Fmoc-Asp(OAI)-OH and Fmoc-Lys(Aloc)-OH, respectively. Allyl functions are removed by treatment with Pd(PPH3)4, morphollne and triphenylphosphine in dry THF at room temperature and cyclization is achieved with PYBOP. Finally the peptide is deblocked and removed from the resin as described in General Procedures A and B.

[0087] In yet another example, the head to tail cyclic peptide TSLLD (SEQ ID NO: 62) can be assembled on Fmoc-Asp(OPAC-resin)-OAI using Fmoc-amino acids and t-butyl side-chain protection, as described in General Procedures A and B in Example 1. The allyl function is removed by treatment with Pd(PPh3)4, morpholine and triphenylphosphine in dry THF at room temperature and cyclization is achieved with PYBOP. Finally the peptide is deblocked and removed from the resin as described in General Procedures A and P to give cyclic LDTSL peptide. Kates, S. A., eL al., “Peptides: Design, Synthesis and Biological Activity,” Basava C, Anantharamalah GM, eds., pp. 39-58 Boston: Birkhauser.

METHODS

[0088] Peptides which mimic the conserved amino acid mocif LDTSL (SEQ ID NO: 1) and which are modified at the N- and C-termini, including the compounds of the present invention, are useful in a method of inhibiting (e.g., reducing or preventing) the binding of a cell bearing a ligand of MAdCAM-1 on the cell surface to MAdCAM-1 or a portion thereof (e.g., the extracellular domain). According to the method, the cell bearing a ligand for MAdCAM-1 is contacted with an effective amount of an (i.e., one or more) inhibitor (as represented by Structural Formula (I) or (II)). As used herein, an inhibitor is a compound which inhibits (reduces or prevents) the binding of MAdCAM-1 to a ligand, including &agr;4&bgr;7 integrin, and/or which inhibits the triggering of a cellular response mediated by the ligand. An effective amount can be an inhibitory amount (such as an amount sufficient to achieve inhibition of adhesion of a cell bearing a MAdCAM-1 ligand to MAdCAM-1). Ligands for MAdCAM-1 include &agr;4&bgr;7 integrins, such as human &agr;4&bgr;7 integrin, and its homologs from other species such as mice (also referred to as &agr;4&bgr;p or LPAM-1 in mice).

[0089] For example, the adhesion of a cell which naturally expresses a ligand for MAdCAM-1, such as a leukocyte (e.g., g lymphocyte, T lymphocyte) or another cell which expresses a ligand for MAdCAM-1 (e.g., a recombinant cell), to MAdCAM-1 can be inhibited in vivo or in vitro according to the method. In one embodiment, the MAdCAM-mediated interaction of a first cell bearing a ligand for MAdCAM-1 with a second cell bearing MAdCAM-1 is inhibited by contacting the first cell with an inhibitor according to the method.

[0090] The adhesion of cells to MAdCAM-1 or a suitable portion thereof can be inhibited. For example, MAdCAM-1 or a suitable portion thereof can be a soluble protein or can be expressed on the surface of a suitable cell, such as a cell which naturally expresses MAdCAM-1 (e.g., an endothelial cell), a suitable cell line, or another cell which expresses MAdCAM-1 (e.g., a recombinant cell). Suitable portions of MAdCAM-1 include, for example, a portion comprising the LDTSL (SEQ ID NO: 1) motif capable of mediating adhesion (e.g., a portion comprising the entire extracellular domain, or both N-terminal immunoglobulin-like domains) (see e.g., Briskin, et al., WO 96/24673, published Aug. 15, 19961). MAdCAM-1 or a portion thereof can be part of a larger molecule, such as a fusion protein. For example, a soluble hybrid protein comprising a mammalian (e.g., human or other primate, murine) MAdCAM-1 moiety fused at its C-terminus, to the N-terminus of an immunoglobulin moiety (e.g., one or more immunoglobulin constant regions) to obtain an immunoadhesin, such as those prepared according to Capon eL al. (U.S. Pat. No. 5,428,130), can be used. For example, as shown herein, the adhesion of a T cell lymphoma line which expresses &agr;4&bgr;7 to a fusion protein comprising the extracellular domain of murine MAdCAM-1 joined to the constant region of a murine K light chain was inhibited according to the method (See e.g., Example 2 and Table 2). The use of a fusion protein comprising the extracellular domain of human MAdCAM-1 fused to a human 51 constant regions is described in Example 3. These or other recombinant soluble receptor molecules are useful in the method.

[0091] In another aspect, the invention relates to a method of treating an individual (e.g., a mammal, such as a human or other primate) suffering from a disease associated with leukocyte (e.g., lymphocyte, monocyte) infiltration of tissues (including recruitment and/or accumulation of leukocytes in tissues) which express the molecule MAdCAM-1. The method comprises administering to the individual a therapeutically effective amount of an inhibitor (i.e., one or more inhibitors) of Structural Formula (I) or Structural Formula (II). For example, inflammatory diseases, including diseases which are associated with leukocyte infiltration of the gastrointestinal tract (including gut-associated endothelium), other mucosal tissues, or tissues expressing the molecule MAdCAM-1 (e.g., gut-assocIated tissues, such as venules of the lamina propria of the small and large intestine; and mammary gland (e.g., lactating mammary gland)) , can be treated according to the present method. Similarly, an individual suffering from a disease associated with leukocyte infiltration of tissues as a result of binding of leukocytes to cells (e.g., endothelial cells) expressing the molecule MAdCAM-1 can be treated according to the present invention.

[0092] Diseases which can be treated accordingly include inflammatory bowel disease (IBD), such as ulcerative colitis, Crohn's disease, ileitis, Celiac disease, nontropical Sprue, enteropathy associated with seronegative arthropathies, microscopic or collagenous colitis, eoslnophilic gastroenteritis, or pouchitis resulting after proctocolectomy, and ileoanal anastomosis.

[0093] Pancreatitis and insulin-dependent diabetes mellitus are other diseases which can be treated using the present method. It has been reported that MAdCAM-1 is expressed by some vessels in the exocrine pancreas from NOD (nonobese diabetic) mice, as well as from BALB/c and SJL mice. Expression of MAdCAM-1 was reportedly induced on endothelum in inflamed islets of the pancreas of the NOD mouse, and MAdCAM-1 was the predominant addressin expressed by NOD islet endothelium at early stages of insulitis (Hanninen, A., et al., J. Clin. Invest., 92: 2509-2515 (1993)). Further, accumulation of lymphocytes expressing &agr;4&bgr;7 within islets was observed, and MAdCAM-1 was implicated in the binding of lymphoma cells via &agr;4&bgr;7 to vessels from inflamed islets (Hanninen, A., et al., J. Clin, Invest., 92: 2509-2515 (1993)).

[0094] Examples of inflammatory diseases associated with mucosal tissues which can be treated according to the present method include mastitis (mammary gland), cholecystitis, cholangitis or pericholangitis (bile duct and surrounding tissue of the liver), chronic bronchitis, chronic sinusitis, asthma, and graft versus host disease (e.g., in the gastrointestinal tract). As seen in Crohn's disease, inflammation often extends beyond the mucosal surface, accordingly chronic inflammatory diseases of the lung which result in interstitial fibrosis, such as hypersensitivity pneumonitis, collagen diseases, sarcoidosls, and other idiopathic conditions can be amenable to treatment.

[0095] Some studies have suggested that the cell adhesion molecule, ICAM-1, can mediate leukocyte recruitment to inflammatory sites through adhesion to leukocyte surface ligands, i.e. , Mac-1, LFA-1 or 94 l (Springer, Nature, 346:425-434 (1990)). In addition, vascular cell adhesion molecule-l (VCAM-1), which recognizes the &agr;4&bgr;1 integrin (VLA-4), has been reported to play a role in in vivo leukocyte recruitment (Silber et al., J. Clin. Invest. 93:1554-1563 (1994)). It has been proposed that IBD can be treated by blocking the interaction of ICAM-1 with LFA-1 or Mac-1, or of VCAM-1 with &agr;4&bgr;1 (e.g., WO 93/15764). However, these therapeutic targets are likely to be involved in inflammatory processes in multiple organs, and a functional blockade could cause systemic immune dysfunction. in contrast to VCAM-1 and ICAM-1, MAdCAM-1 is preferentially expressed in the gastrointestinal tract and mucosal tissues, binds the &agr;4&bgr;7 integrin found on lymphocytes, and participates in the homing of these cells to mucosal sites, such as Peyer's patches in the intestinal wall (Hamann et al., J. Immunol., 152:3282-3293 (1994)). As inhibitors of the binding of MAdCAM-1 to &agr;4&bgr;7 integrin, the compounds of the present invention have the potential for fewer side effects due to e.g., effects on other tissue types where adhesion is mediated by other receptors, such as &agr;4&bgr;1 integrin.

[0096] According to the method, an inhibitor can be administered to an individual (e.g., a human) alone or in conjunction with another agent, such as an additional pharmacologically active agent (e.g., sulfasalazine, an antiinflammatory compound, or a steroidal or other non-steroidal antiinflammatory compound). A compound can be administered before, along with or subsequent to administration of the additional agent, in amounts sufficient to reduce or prevent MAdCAM-mediated binding to a ligand for YAdCAM-1, such as human &agr;4&bgr;7.

[0097] An effective amount of an inhibitor can be administered by an appropriate route in a single dose or multiple doses. An effective amount is a therapeutically effective amount sufficient to achieve the desired therapeutic and/or prophylactic effect (such as an amount sufficient to reduce or prevent MAdCAM-mediated binding to a MAdCAM ligand, thereby inhibiting leukocyte adhesion and infiltration and associated cellular responses. Suitable dosages of inhibitors of Structural Formula (I) or (II) for use in therapy, diagnosis or prophylaxis, can be determined by methods known in the art and can be dependent, for example, upon the individual's age, sensitivity, tolerance and overall well-being. For example, dosages can be from about 0.1 mg/kg to about 50 mg,/kg body weight per treatment.

[0098] A variety of routes of administration are possible including, but not necessarily limited to parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous injection), oral (e.g., dietary), topical, inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops), or rectal, depending on the disease or condition to be treated. Oral and parenteral administration are preferred modes of administration.

[0099] Formulation of an inhibitor to be administered will vary according to the route of administration selected (e.g., solution, emulsion, capsule). An appropriate composition comprising the compound to be administered can be prepared in a physiologically acceptable vehicle or carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. (1980)). For inhalation, the compound can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer, nebulizer or pressurized aerosol dispenser).

[0100] The present method can be used to assess the inhibitory effect of a compound of the present invention and of other potential antagonists useful in the method on the interaction of MAdCAM-1 with a ligand for MAdCAM-1 in vitro or in vivo. For example, compounds of the present invention were assayed for their ability to inhibit MAdCAM-1 binding to human &agr;4&bgr;7 integrin using an adhesion assay described in Example 2 and Example 3. Other suitable assays can be used to assess the ability of compounds to inhibit binding of MAdCAM-1 to a ligand for MAdCAM-1. For example, other fusion proteins (e.g., a chimeric protein or “immunoadhesin”) can be constructed and used in an assay such as the assay described in Example 2 or other suitable assays. A fusion protein comprising human MAdCAM-1 or a portion thereof (e.g., the entire extracellular domain or the two N-terminal immunoglobulin domains) joined to an immunoglobulin heavy or light chain constant region can be produced and used in an assay similar to that described in Example 2, using a capture antibody suitable for the immunoglobulin constant region selected. In a different assay, unlabeled cells bearing a MAdCAM-1 ligand can be contacted with such a fusion protein under conditions suitable for binding to the ligand in the presence or absence of compound, and the amount of bound chimeric protein determined (e.g., cells can be removed and the amount of chimeric protein bound determined by, flow cytometry or other suitable methods)

[0101] Compounds suitable for use in therapy can also be evaluated in vivo, using suitable animal models. Suitable animal models of inflammation have been described. For example, NOD mice provide an animal model of insulin-dependent diabetes mellitus. CD45 RBH-/SCID model provides a model in mice with similarity to both Crohn's disease and ulcerative colitis (Powrie, F. et al., Immunity, 1: 553-562 (1994)). Captive cotton-top tamarins, a New World nonhman primate species, develop spontaneous, often chronic, colitis that clinically and histolgocially resembles ulcerative colitis in humans (Madara, J. L. et al., Gastroenterology, 88: 13-19 (1985)) The tamarin model and other animal models of gastrointestinal inflammation using BALB/c mice (a (DSS)-induced inflammation model; DSS, dextran sodium sulfate) and common marmosets are described in Briskin et al., U.S. Ser. No. 08/523,004, filed Sep. 1, 1995, the teachings of which are incorporated herein by reference in their entirety (see also Briskin et al., WO 96/24673, published Aug. 15, 1996). Knockout mice which develop intestinal lesions similar to those of human inflammatory bowel disease have also been described (Strober, W. and Ehrhardt, R..O., Cell, 75: 203-205 (1993)).

[0102] Preferably, selective inhibition of the interaction of MAdCAM-1 with a ligand thereof is achieved. Selective inhibition can be assessed, for example, by further evaluating the effect of compounds on adhesion between one or more other receptor-ligand pairs. For example, adhesion assays which assess the interaction of a particular receptor and ligand pair, such as (a) VCAM-1 and &agr;4&bgr;1, (b) ICAM-1 and LFA-1, (c) fibronectin and &agr;5&bgr;1, and (d) fibronectin and &agr;4&bgr;1, can be conducted. Nonlimiting examples of suitable cell lines for such adhesion assays include (a) HAMOS cells, (b) JY cells, (c) K562 cells, and (d) RAMOS cells, respectively. In these assays, isolated and/or recombinant fibronectin (Sigma Chemical Co., St. Louis, Mo.), VCAM-1, ICAM-1, or fusion proteins comprising the ligand binding domain(s) of VCAM-1 or ICAM-1, can be used, for example.

[0103] The compounds of the present invention can also be used as immunogens to produce antibodies, including monoclonal and polyclonal antibodies, against MAdCAM-1 using methods known in the art or other suitable methods (see e.g., Kohler et al., Nature, 256:495-497 (1975); Galfre, G., et al., Nature, 299:550-552 (1977); Harlow et al., 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor, N.Y.); or Current Protocols in Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel et al., Eds. (John Wiley & Sons: New York, N.Y.), Chapter 11 (1991)). Antibodies can be raised against an appropriate immunogen in a suitable mammal (e.g., a mouse, rat, rabbit or sheep) For example, a compound represented by Structural Formulas (I) and (II) or a variant thereof can be produced and used as an immunogen to raise antibodies in a suitable immunization protocol.

[0104] Antibody-producing cells (e.g., a lymphocyte) can be isolated from, for example, the lymph nodes or spleen of an immunized animal. The cells can then be fused to a suitable immortalized cell (e.g., a myeloma cell line), thereby forming a hybridoma. Fused cells can be isolated employing selective culturing techniques. Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).

[0105] Antibodies and monoclonal antibodies produced as described have a variety of uses. For example, those against or reactive with MAdCAM-1, and preferably which bind specifically to MAdCAM-1, can be used to identify and/or sort cells exhibiting MAdCAM-1 on the cell surface (e.g., in fluorescence activated cell sorting, histological analyses) Monoclonal antibodies specific for MAdCAM-1 can also be used to detect and/or quantitate MAdCAM-1 expressed on the surface of a cell or present in a sample (e.g., in an ELISA). Antibodies reactive with the immunogen are also useful. For example, they can be used to detect and/or quantitate immunogen in a sample, or to purify immunogen (e.g., by immunoaffinity purification).

[0106] This invention further relates to the diagnostic use or research use of antagonists of MAdCAM in the detection and/or quantitation of &agr;4&bgr;7 integrin present on leukocytes using a suitable label or indicator. For example, hydrocarbon fluorescence indicators such as a pyrene moiety, or radioisotope labels such as 125I can be attached to the aryl ring of the N-terminus functional group of the inhibitor or antagonist peptide of this invention. The fluorescence properties of pyrene ring derivatives have been reported (I. A. Prokhorenko, et al., Bioorg. Med. Chem. Lett., 5:2081 (1995)). The use of labeled peptides for identifying cells having a membrane-bound protein is described in Riper et al., J. Exp. Med. 177:851 (1993).

[0107] This diagnostic tool would be valuable in the identification of &agr;4&bgr;7-positive leukocytes from subjects with suspected inflammatory bowel diseases and the like. Antagonist molecules of this invention possessing this indicator grouping are described as examples of the invention with adhesion antagonist properties.

EXEMPLIFICATION

[0108] The present invention will now be illustrated by the following Examples, which are not intended to be limiting in any way.

Example 1: Synthesis

[0109] The novel compounds of this invention can be synthesized according to the general procedures of synthesis, A-D, utilizing solid-phase peptide synthesis methodology described herein, which are known to person skilled in the art, or other suitable techniques. See e.g., Merrifield, R. B., Science, 232: 341 (1980); Carpino, L. A., Han, G. Y., J. Org. Chem., 37: 3404 (1972); Gauspohl, H., et al., Synthesis, 5: 315 (1992)).

[0110] For multiple peptlde synthesis the Fmoc/t-Bu protocol was used. In situ activation of the amino acid derivative was performed by benzotriazolyl-N-oxytripyrrolidinophosphoniumhexaflurophosphate (PYBOP) or 2(1-benzotriazolyl-1-yl)-1,1,3,3-tetramethyluronium (HBTU) and N-methylmorpholine (NMM) in dimethylformamide (DMF) as a solvent. In order to improve the coupling efficiency and quality of the final peptides, each coupling reaction was carried out twice. As a solid support Rink Amide Am Resin (4-[2′-4-dimethoxyphenyl-Fmoc-aminomethyl]-phenoxyacetamido-norleucylaminoethyl resin) was used due to its high loading and excellent swelling properties. Fmoc group was removed by 10% piperidine and 2% DBU in DMF. The peptides were cleaved from the resin and the side chain protecting groups were simultaneously removed by a trifluoroacetic acid (TFA) cocktail. All peptides reported in Table 2 below are carboxy-terminus blocked as the carboxamide. More details of the peptide synthesis protocol are given below.

[0111] HPLC and Mass Spectral Analysis

[0112] All of the crude peptides were analyzed by reversed phase HPLC using DELTAPAK C18, 5 &mgr;m column, eluted with a linear gradient of 0.1% TFA in CH3CN/water (100% CH3CN/0% water) to 0.1% TPA in CH3CN/water (0% CH3CN/100% water) over a 30 minute period with flow rate of 1 ml/minute. The purity of the samples were determined and were essentially found to contain one component. This was confirmed by matrix-assisted laser desorption ionization time of flight mass spectral analysis (MALDI-TOF, Kratos, Inc.) internally referenced to leucine-enkephalin and sinapinic acid. Generally, the peptides gave the mass within 1%, of MH+ or M+Na+ or within experimental error of the calculated value.

[0113] General Procedure A, Peptide Synthesis

[0114] These peptides were synthesized via Fmoc/t-Butyl chemistry on a Gilson 421 automated multiple peptide synthesizer starting with Fmoc-Am-PS (100 mg, 0.5 mmol/g) resin. Acylations were carried out twice with the Fmoc-amino acid (5 equivalents) , HDTU (5 equivalents) /NMM (10 equivalents) using 20-45 minute coupling time. Fmoc deprotection was carried out with 20% piperidine in DMF for 24 minutes. Treatment with Reagent-R (TFA-EDT-thioanisole-anisole, 90:5:3:2) for 2 hours was used to deblock and remove the peptides from the resin. The peptides were then precipitated from ether and lyophilized from acetic acid.

[0115] General Procedure B, Peptide Synthesis

[0116] Peptides were synthesized via Fmoc/t-Butyl chemistry on a Gilson 421 automated multiple peptide synthesizer starting with Fmoc-Am-PS (50 mg, 0.5 mmol/g) resin. Acylations were carried out twice with the Fmoc-amino acid (10 equivalents), HBTU (10 equivalents)/NMM (20 equivalents) or PYBOP (10 equivalents)/NMM (20 equivalents) using a 20-45 minute coupling time. Fmoc deprotection was carried out with 2% 1,8-diazobicyclo [5,4.0]undec-7-ene (DBU) and 10% piperidine in DMF for 24 minutes. Treatment with Reagent-R (TFA-EDT-thioanlsole-anisole, 90:5:3:2) for 2 hours was used to deblock and remove the peptides from the resin. The peptides were then precipitated from ether and lyophilized from acetic acid.

[0117] General Procedure C, Synthesis of N-Acylated Peptides

[0118] The peptides were synthesized via Fmoc/t-Butyl chemistry on a Gison 421 automated multiple peptide synthesizer starting with Fmoc-Am-PS (50 mg, 0.5 mmol/g) resin. Acylations were carried out twice with the Fmoc-amino acid (10 equivalents), HETU (10 equivalents)/NMM or PYBOP (10 equivalents)/NMM (20 equivalents) using 20-45 minute coupling time. For the final acylation the Fmoc-amino acid was substituted with an appropriate organic acid. Fmoc deprotection was carried out with 20 DBU and 10% piperidine in DMF for 24 minutes. Treatment with Reagent-R (TFA-EDT-thioanisole-anisole, 90:5:3:2) for 2 hours was used to deblock and remove the peptides from the resin. The peptides were then precipitated from ether and lyophilized from acetic acid.

[0119] General Procedure D, Synthesis Peptides Cyclized Through A Disulfide Bond

[0120] The linear acyclic peptides were synthesized as described above in the General Procedure B. The acetamidomethyl (Acm) protecting group from the cysteine side chain was simultaneously removed and peptides were cyclized by iodine treatment. A solution of 25 mg of iodine in 5 mL of 80% aqueous acetic acid was added to 5 mg of peptide. The mixture was shaken at room temperature for 2 hours, diluted with water (25 ml), extracted with chloroform (3×25 ml) and finally lyophilized to give the cyclized peptide.

Example 2: Biological Activity

[0121] The compounds of the present invention were evaluated for their biological properties. Table 2 lists a number of compounds of the present invention, their physical characterization, and their ability to inhibit the adhesion of &agr;4&bgr;7-bearing cells (HUT 78 cells) to MAdCAM-1 (described as percent inhibition at the concentration noted or by the corresponding IC50 value (am) determined using the adhesion assay described below. IC50 corresponds to the concentration of compound which inhibits 50% of the total number of cells adhering to MAdCAM-1 in a control conducted in the absence of inhibitor.

[0122] Overview

[0123] A soluble fusion protein comprising murine MAdCAM-1 was produced in a baculovirus expression system and used in the adhesion assay. A fusion gene in which sequences encoding the signal peptide and extracellular domain of murine MAdCAM-1 were fused to sequences encoding a constant region of the murine kappa light chain (mCk) was constructed, and cloned into a baculovirus shuttle vector. Fusion protein produced from the resulting construct contained the integrin binding sequences of murine MAdCAM-1 at the amino-terminus, and the mCk sequence at the carboxy-terminus. Recombinant baculovirus encoding the fusion protein was harvested from infected Sf9 insect cells. Fusion protein was detected in supernatants of infected Sf9 cells by ELISA assay, using a horseradish peroxidase-linked polyclonal anti-mCk antibody and chromogenic substrate according to standard protocols. Recombinant protein was also verified by immunoprecipitation with anti-murine MAdCAM-1 monoclonal antibody (MAb MECA-367).

[0124] For adhesion assays, a dose response curve obtained using increasing amounts of rat anti-mCk MAb indicated the amount of rat anti-mCk MAb to be used as capture antibody in the assay. Subsequently, an IC50 for Ac-LDTSL-NH2 was determined to be 278 &mgr;M.

[0125] Human T-cell lymphoma cells (HUT 78 cells) activated with Mn+2 were used for the adhesion assay in a 96-well format. HUT 78 cells were labelled by preincubation with ECECF-AM stain (Molecular Probes). Assays were conducted in a final volume of 200 &mgr;l. The adhesion of HUT 78 cells to MAdCAM-1/mCk fusion protein bound to wells via rat anti-mCk capture antibody was assessed in the presence or absence of each compound. Adhesion of HUT 78 cells was monitored using a fluorescent plate reader at a setting gain of 10 at 485/535 nM. Percent inhibition and IC50 values were determined.

[0126] Production of MAdCAM-1/mCk Fusion Protein Antibodies, Cells and Viruses

[0127] Affinity-purifled polyclonal goat-anti-mouse-C-kappa (mCk) antibodies (#M33100), horseradish peroxidase-linked goat anti-mCk antibodies (#M33107), and alkaline phosphatase-linked swine anti-goat H & L chains (#G50008) were purchased from Caltag. Sepharose-linked rat anti-mCk affinity matrix was obtained from Zymed.

[0128] Affinity purified rat-anti-mCk monoclonal antibodies were prepared by Dr. Hans Peter Kocher (BTC) from the rat hybridoma cell line 187.1 (American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209, Accession No. ATCC HB 58), and were linked to cyanogen bromide-activated Sepharose 4B for affinity purification of proteins.

[0129] ACMNPV DNA and cationic liposomes (Invitrogen transfection kit #B825-03) or lipofectin (GIBCO/BRL #82925A) were used to transfect Sf9 insect cells (American Type Culture Collection, Accession No. ATCC CRL 1711; gift from Dr. Max Summers) in HyQ serum-free medium (Hyclone, #B-5502-L-P). BaculoGold (Pharmingen) was also used in some transfections. Cells were maintained in TNM-FH medium (GIBCO BRL) supplemented with 10% fetal bovine serum and 0.1% F-68 pluronic acid (GIBCO/BRL, #670-404AG).

[0130] Construction of pVL941/mCk

[0131] Based on the published sequence for the mouse kappa constant region (mCk; Hieter, P. A., et al., Cell 22: 197-207 (1980)), two oligonucleotides having the following sequences (SEQ ID NO: 5 and SEQ ID NO: 6, respectively) were designed and synthesized: 1 5′ primer:     5′-GGATCC GCT GAT GCT GCA CCA ACT GTA TTC-3′ 3′ primer:     5′-CCT TTG TCC TAA CAC TCA TTC CTG TT-3′

[0132] The mCk coding sequence was amplified by polymerase chain reaction (PCR) from the plasmid pVL91A3 containing the full-length 91A3 mouse kappa light chain (Meek, K., et al., Proc. Natl. Acad. Sci. U.S.A., 84: 6244-6248 (1987)) An in-frame BamHI restriction site (underlined in the sequence above) was incorporated at the 5′-end of the coding sequence to facilitate fusion with MAdCAM-1 sequences.

[0133] Vector pVL941 (Invitrogen) was linearized by digestion at she unique BamHI site in the polyhedrin gene, and the ends were made blunt with the Klenow fraqment of DNA polymerase. The amplified DNA fragment (312 base pairs) containing the 91A3 mouse kappa light chain was then ligated to pVL941 to yield pVL941/mCk. Clones containing the mCk sequence in the same 5′ to 3′ orientation as the polyhedrin promoter were selected.

[0134] Construction of Baculovirus Shuttle Vector

[0135] Based on the published nucleotlde sequence of murine MAdCAM-1 (Briskin, M. J. et al., Nature, 363: 461-464 (1993)), oligonucleotide primers having the following sequences were designed and synthesized: 2 5′ primer:     5′-GAT CAG GGA TCC ATG GAA TCC ATC CTG GCC CTC CTG-3′ 3′ primer:     5′-TCG ATC GGA TCC GGT GGA GGA GGA ATT CGG GGT CA-3′

[0136] The ATG start codon of MAdCAM-1 is indicated in bold. The 5′ and 3′ primers each incorporated a PamHI restriction site for cloning into the expression vector (underlined above). Murine MAdCAM-1 sequences were amplified by PCR using these 5′- and 3′-primers. The product was digested with BamEI and inserted into vector pVL941/mCk, which had been digested with BamHI. DNA fragments cloned into the BamHI site were screened for the correct orientation with respect to the mCk sequence. Fusion proteins produced from the resulting construct contained mouse MAdCAM-1 sequences at the N-terminus, and the mCk sequence at the carboxy-terminus (FIG. 3). A two-amino acid glycine-serine spacer encoded by the six nucleotides corresponding to the BamHI restriction site forms the junction between these sequences.

[0137] Transfection of Sf9 Insect Cells

[0138] Co-transfection (lipofection) of Sf9 insect cells with a mixture of 1 &mgr;g of ACMNPV DNA (Invitrogen), 3-5 &mgr;g of shuttle vector DNA (pVL941/MAdCAM-1/mCk purified by magic-mini prep, Promega) and cationic liposomes (30 &mgr;l) was performed according to the manufacturers' instructions, with slight modifications. 2×106 &mgr; cells in TNM-FH medium (10% FBS) were plated out in 60 mm dishes and allowed to attach over a 2-4 hour period prior to transfection. The medium was removed and the adherent cells were washed twice with serum-free medium (HyQ). Three ml of HyQ containing the DNA/liposome mixture were applied dropwise over the cells; the cells were incubated overnight. The medium was then removed by aspiration and 5 ml TNF-FN medium containing 106 FBS was added to each dish. After 48 hours, one ml was removed for plaque purification of recombinant virus, as previously described (O'Reilly, D. R., et al., (Eds.) Baculovirus Expression Vectors (W. H. Freeman and Co.: New York), pp. 124-128 (1992)).

[0139] ELISA Assav of Transfected Sf9 Cells For each transfLection, triplicate wells were coated with polyclonal goat anti-mCk antibodies (2 &mgr;g/ml in carbonate/bicarbonate buffer), and incubated overnight. The plates were washed three times with phosphate buffered saline (PBS) containing 0.5% Triton X-100, and 0.5 M NaCl and then washed twice with PBS. This washing protocol was used between all steps. The wells were then blocked with 2% BSA in TBS for one hour. Supernatant (100 &mgr;l) taken from cells four to five days post-infection was aoplied to each well and incubated at 37° C. for 2 hours. The presence of mCk fusion protein was detected by the addition of horseradish peroxidase-linked polyclonal anti-mCk antibody and chromogenic substrate, according to standard protocols.

[0140] Immunodetection of mCk fusion proteins Recombinant virus containing the MAdCAM-1/mCk fusion gene was isolated by plaque purification, and amplified by infecting Sf9 insect cells (2×106 cells/60 mm petri dish) in 5 ml TNM-FH medium. From this 5 ml stock, an aliquot (100 &mgr;l) was taken to infect 2×106 Sf9 cells as before. Supernatants (10 &mgr;l) taken 24, 48, 72 and 96 hours post-infection were assayed for the presence, accumulation and stability of mC, fusion protein by Western blot analysis. Samples were applied to 10% SDS/PAGE gels. Proteins were electrophoretically transferred to nitrocellulose by standard techniques. The fusion protein was detected by addition of polyclonal goat anti-mCkantibodies (2 &mgr;g) in 5 ml BLOTTO (5%) followed by treatment with alkaline phosphatase-linked swine anti-goat H & L chains and chromogenic substrate (BIORAD, Catalog No. 170-6432).

[0141] Purification of mCk Fusion Protein by Affinity Chromatography

[0142] Production of fusion proteins was carried out in stirred microcarrer flasks. Sf9 insect cells (2×106 cells/ml) were infected with virus particles containing the MAdCAM-1/mCkfusion gene at a multiplicity of infection of 0.001. Upon complete lysis of the cells (approximately eight days later), the spent medium was clarified by low steed centrifugation. The supernatant was applied to a rat monoclonal antli-mCk-coupled Sepharose column (purchased from Zymed or prepared usina rat monoclonal 187.1) equilibrated in TBS pH 7.0. The column was washed with TBS pH 7.0, and the bound mCk fusion protein eluted in glycine buffer pH 2.2 and neutralized by the addition of 2M Tris pH 8.0. Yields of 2 mg of mCk fusion protein per liter of cells were obtained.

[0143] Results

[0144] MAdCAM-1/mCk fusion protein was expressed under the transcriptional regulation of the viral polyhedrin promoter. To transfer the chimeric gene into the baculovirus genome, pVL941/MAdCAM-1/mCk plasmid was co-transfected with A. californica nuclear polyhedrosis virus DNA into Sf9 insect cells. Several recombinant plaques were isolated and one was chosen for more detailed characterization.

[0145] ELISA assays on Transfected Cell Supernatants

[0146] To determine if an ELISA assay could be used to detect the appearance of fusion protein in the supernatant of transfected cells, aliquots (500 &mgr;l) were taken from the supernatant of transfected cells at 3, 4 and 5 days post-transfection, and ELISA assays were performed as described above. The results indicated that the sensitivity of the ELISA assay was sufficient to detect the mCk fusion protein. The assay can be used to provide an indication that the desired gene is expressed in the insect cells. Moreover, a positive signal in the ELISA assay correlated well with the isolation of recombinant viral particles in subsequent plaque assays.

[0147] Cellular Adhesion Assay

[0148] The Following buffers and reagents were used in the initial titration of capture antibody and in the adhesion inhibition assays: 3 TABLE 1 CARBONATE BUFFER: 17.2 g NaHCO3 Sigma #S-5575 8.6 g Na2CO3 Sigma #S-6139 Bring volume to 1L with H20 1% BSA/PBS BSA 5 g Sigma #A-6793 PBS 500 ml GIBCO #14040-026 Sterile filter ASSAY BUFFER (HBSS/2% FCS/ 25mM HEPES/ Pen/Strep., pH 7.2): HBSS 500 ml Gibco #14025-02 FCS 10 ml Gibco #16000-044 HEPES (1M) 12.5 ml Gibco #15630-015 Pen/Strep. (100X) 5 ml Gibco #15070-022 Sterile filter 2X ASSAY BUFFER (2X HBSS/4%FCS/ 50mM HEPES/ Pen/Strep., pH 7.2): 10x HBSS 100 ml Gibco #14065-023 FCS 20 ml Gibco #16000-044 HEPES (1M) 25 ml Gibco #15630-015 NaHCO3(7.5%) 4.7 ml Gibco #25080 Pen/Strep. 10 ml Gibco #15070-022 Check pH Bring volume up to 500 ml with H2O Sterile filter BCECF-AM Molecular #3-1170 Probes DMSO Sigma #D-8779

[0149] Maintenance and Labelling of Cells

[0150] HUT 78 cells (a human T cell lymphoma line; American Type Culture Collection, Accession No. ATCC TIB 161) were maintained in culture for up to one month in RPMI 1640 supplemented with 10% FCS, 20 mM HEPES, and Pen-Strep (1:100). Just prior to use in adhesion assays, cells were labeled with BCECF-AM as follows: 1×107 cells were pelleted at 1000 rpm for 10 minutes and resuspended in 25 mL cold PBS (Phosphate Buffered Saline). The cells were pelleted again, resuspended in cold PBS, and pelleted again. The cell pellet was resuspended in 25 ml PBS and labeled with 50 &mgr;l BCECF-AM (1 &mgr;g/&mgr;L in DMSO) for 30 minutes at 37° C. (BCECF-AM; 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyflourescein, acetoxymethyl ester). The labeled cells were pelleted at 700 rpm for 10 minutes, resuspended in 25 ml cold Assay Buffer and pelleted. Finally, labeled cells were counted and resuspended in Assay Buffer (ambient temperature) at a concentration of 2.5×106/ml.

[0151] Titration of Capture Antibody

[0152] A rat anti-mCk antibody (affinity purified rat-anti-mCk monoclonal antibodies from rat hybridoma cell line 187.1; ATCC Accession No. HB58) was selected for use as capture antibody in the adhesion assay. A titration experiment was conducted in order to determine the optimal capture antibody concentration. Assay Buffer for the titration was 2X Assay Buffer.

[0153] ELISA plates were coated with various concentrations capture antibody by adding 10 &mgr;g/ml, 5 &mgr;g/ml, 2.5 &mgr;g/ml, or 1.25 &mgr;g/ml of antibody in a 50 &mgr;l volume to each well and incubating at 4° C. overnight. The next day, plates were blocked with 100 &mgr;l 1% BSA/PBS at 37° C. for 1 hour. After one hour, the BSA/PBS solution was removed, and 50 &mgr;l MAdCAM-mCk fusion protein (neat supernatant) was added to each well and incubated for 1 hour at 37° C.

[0154] Anti-murine MAdCAM-1 antibody MECA-367 (American Type Culture Collection, Accession No. ATCC HB 9478) was used as a blocking antibody. For the titration, either 20 &mgr;l of Assay Buffer (i.e., 2X Assay Buffer), 20 &mgr;l of neat supernatant containing anti-MAdCAM-1 MECA-367 antibody, or 20 &mgr;l of neat supernatant containing MECA-367 diluted 1:2, 1:4, 1:8 or 1:16 in Assay Buffer, was added to each well. The undiluted supernatant contained approximately 3 &mgr;g/ml of antibody.

[0155] Frozen BCECF-labeled HUT 78 cells were thawed and resuspended in Assay Buffer at 2.5×106 cells/ml. 50 &mgr;l of cells, 50 &mgr;l of Assay Buffer (without Pen/Strep), 30 &mgr;l of water, 50 &mgr;l of 8 mM MnCl2, and 20 &mgr;l of either MECA-367 antibody (neat or diluted) or 20 &mgr;l of Assay Buffer alone, were then added to each well, and the plates were incubated on a rotator for 30 minutes at room temperature.

[0156] After incubation, a baseline measurement for each well of total fluorescence was taken using a Fluorescence Concentration Analyzer (IDEXX) at 485/535 nM. Then plates were washed twice with a solution of 50 mM Tris/2 mM MnCl. using an EL 404 Microplate Autowasher (BIO-TEK Instruments) , and fluorescence (due to adherent cells) was determined again using a Fluorescence Concentration Analyzer (IDEXX) at 485/535 nM. For each well, the final reading was divided by the baseline reading. The values from duplicate wells were averaged, and plotted (FIG. 1). Accordingly, 50 &mgr;l of a 5 &mgr;g/mL solution of rat anti-mCk was used to coat each well for subsequent adhesion assays.

[0157] Inhibition Assays and IC50 Determination

[0158] The assay was performed in a 96-well format. Plates were coated overnight at 4° C. with 53 &mgr;l/well of a 5 &mgr;g/mL solution of rat anti-mCk in carbonate buffer. The plates were blocked for 1 hour at 37° C. with 100 &mgr;L/well of 1% BSA in PBS. The BSA/PBS solution was removed, and 50 &mgr;L of MAdCAM-mCk supernatant was added neat to each well. The assay was conducted under conditions in which capture antibody was the limiting reagent.

[0159] Cell adhesion was measured in the presence or absence of compounds. The compounds were resuspended in 100% DMSO, subsequently diluted 1:10 in water, and finally diluted again 1:10 in the assay: 50 &mgr;L of 2X Assay Bufler, 30 &mgr;L water, 20 &mgr;L of compound were added to each well. 50 &mgr;L of a cell suspension containing BCECF-AM labeled HUT 78 cells (resuspended in assay buffer at a concentration of 2.5×106/ml; see above) were added to each well, followed by 50 &mgr;L of a solution of 8 mM MnCl2 in assay buffer. Cell adhesion occurred in 30 minutes at ambient temperature, after which plates were washed with 50 mM Tris/2 mM MnCl2, pH 7.2 using an EL 404 Microplate Autowasher (BIO-TEK Instruments) using the following wash parameters: wash volume=500 &mgr;L; two wash cycles; wash depth=80; aspirating after each wash. Plates were read using a Fluorescence Concentration Analyzer (IDEXX) at 485/535 nM.

[0160] IC50 Determinations

[0161] In order to determine an IC50 (the concentration of compound which inhibits 50% of the total number of cells adhering to MAdCAM-1 relative to a control conducted in the absence of inhibitor), compounds were tested for inhibition of HUT 78 cell adhesion as described above at various concentrations ranging between 62.5 to 500 &mgr;M. Additional determinations were conducted as needed, so that each IC50 was determined over a range which encompassed the IC50. Over this range, Four different concentrations were tested in duplicate. For example, compound 793-1a was tested at four different concentrations (50 &mgr;M, 25 &mgr;M, 12.5 &mgr;M and 6.25 &mgr;M) in duplicate wells.

[0162] For each compound, a ratio was determined as follows: An average of the readings from duplicate wells was divided by an average of eight control wells (adhesion in the absence of any compound). Percent inhibition of adhesion was calculated as 100×(1−the ratio). In addition, the concentration (&mgr;M) of compound used was plotted against the resulting ratios. The resulting plot for compound 793-1a is shown in FIG. 2.

[0163] The IC50 was then determined using Kaleidograph software (Abelbeck Software). The complete procedure was repeated again using duplicate wells for each point. An average of two IC50 determinations was obtained, and the resulting values are presented in Table 2 with percent adhesion inhibition for a variety of compounds of the present invention. For example, the IC50 for compound 793-1a was determined to be 14 &mgr;M. Cysteines in cyclic peptides which are linked by a disulfide bond are indicated by an “*”.Abbreviations used are defined in Table 3 below.

[0164] In Table 2, Example No. 1 is represented by SEQ ID NO: 7; Example No. 2 is represented by SEQ ID NO: 8; Example No. 3 is represented by SEQ ID NO: 9; Example No. 4 is represented by SEQ ID NC: 10; Example No. 5 is represented by SEQ ID NO: 11; Example No. 6 is represented by SEQ ID NO: 12; Example No. 7 is represented by SEQ ID NO: 13; Example No. 8 is represented by SEQ ID NO: l4; Example No. 9 is represented by SEQ ID NO: 15; Example No. 10 is represented by SEQ ID NO: 7; Example No. 11 is represented by SEQ ID NO: 16; Example No. 12 is represented by SEQ ID NO: 17; Example No. 13 is represented by SEQ ID NO: 18; Example No. 14 is represented by SEQ ID NO: 9; Example No. 15 is represented by SEQ ID NO: 13; Example No. 16 is represented by SEQ ID NO: 19; Example No. 17 is represented by SEQ ID NO: 20; Example No. 18 is represented by SEQ ID NO: 21; Example No. 19 is represented by SEQ ID NO: 22; Example No. 20 is represented by SEQ ID NO: 23; Example No. 21 is represented by SEQ ID NO: 24; Example No. 22 is represented by SEQ ID NO: 25; Example No. 23 is represented by SEQ ID NO: 26; Example No. 24 is represented by SEQ ID NO: 7; Example No. 25 is represented by SEQ ID NO: 27; Example No. 26 is represented by SEQ ID NO: 28; Example No. 27 is represented by SEQ ID NO: 29; Example No. 28 is represented by SEQ ID NO: 30; Example No. 29 is represented by SEQ ID NO: 31; Example No. 30 is represented by SEQ ID NO: 32; Example No. 31 is represented by SEQ ID NO: 33; Example No. 32 is represented by SEQ ID NO: 34; Example No. 33 is represented by SEQ ID NO: 35; Example No. 34 is represented by SEQ ID NO: 36; Example No. 35 is represented by SEQ ID NO: 37; Example No. 36 is represented by SEQ ID NO: 38; Example No. 37 is represented by SEQ ID NO: 39;

[0165] Example No. 38 is represented by SEQ ID NO: 40; Example No. 39 isple No. 40 is represented by SEQ ID NO: 42; Example No. 41 is r epresented by SEQ ID NO: 43; Exampl e No. 42 is represented by SEQ ID NO: 44; Example No. 43 is represented by SEQ ID NO: 45; Example No. 44 is represented by SEQ ID NO: 46; Example No. 45 is rep resented by SEQ I D NO: 47; Example No. 46 is represented by SEQ ID NO: 48; Example No. 47 is represented by SEQ ID NO: 49; Example No. 48 is represented by SEQ ID NO: 50; Example No. 49 is represented by SEQ ID NO: 51; Example No. 50 is represented by SEQ ID NO: 52; Example No. 51 is represented by SEQ ID NO: 53; Example No. 54 is represented by SEQ ID NO: 54; Example No. 55 is represented by SEQ ID NO: 55; Example No. 56 is represented by SEQ ID NO: 56; Example No. 57 is represented by SEQ ID NO: 57; Example No. 58 is represented by SEQ ID NO: 7; Example No. 59 is represented by SEQ ID NO: 58; Example No. 60 is represented by SEQ ID NO: 59; Example No. 61 is represented by SEQ ID NO: 60; Example No. 62 is represented by SEQ ID NO: 61. 4 TABLE 2 % % Adhesion Method MH + Purity Inhibition IC50 Ex No L# Sequence Prep Cal. Found HPLC 500 &mgr;M (&mgr;M) 1 00112-1A Ac-Leu-Asp-Thr-Ser-Leu B 591 616 75 278 2 00619-1A Ac-Leu-Asp-Ala-Ser-Leu B 557 563 78 47 1004 3 0092-1A Ac-His-Trp-Arg-Gly-Leu-Asp-Thr B 926 926 50 49 584 4 00167-1B Ac-Cys*-Leu-Asp-Thr-Ser-Leu-Cys* B,D 797 816 67 51 (@250 &mgr;M) 102 .4 5 00145-1B Ac-Cys*-Gly-Leu-Asp-Thr-Ser-Leu-Gly-Cys* B,D 909 925 80 45 (@200 &mgr;M) 266 6 0093-1A Ac-Trp-Arg-Gly-Leu-Asp-Thr-Ser B 877 878 42 44 862 7 0096-1A Ac-Trp-Arg-Gly-Leu-Asp-Thr B 789 792 53 46 725 8 0094-1A Ac-Arg-Gly-Leu-Asp-Thr-Ser-Leu B 804 802 34,41 35 1575 9 0099-1A Ac-Gly-Leu-Asp-Thr-Ser-Leu B 648 647 67 58 339 10 0104-1A Ac-Leu-Asp-Thr-Ser-Leu B 591 616 47 50 530 11 0093-1A Ac-Trp-Arg-Gly-Leu-Asp-Thr-Ser B 877 878 42 44 862 12 0097-1A Ac-Arg-Gly-Leu-Asp-Thr-Ser B 691 691 32,34 31 1789 13 0108-1A Ac-Gly-Leu-Asp-Thr-Ser B 535 554 73 43 745 14 0092-1A Ac-His-Trp-Arg-Gly-Leu-Asp-Thr B 926 926 50 49 584 15 0096-1A Ac-Trp-Arg-Gly-Leu-Asp-Thr B 789 792 53 46 725 16 0105-1A Ac-Arg-Gly-Leu-Asp-Thr B 603 607 45 48 542 17 00379-1A Ac-MeLeu-Leu-Asp-Thr-Ser-Leu B 965 966 50 28 1316 18 00380-1A Phx-Leu-Asp-Thr-Ser-Leu B,C 722 722 32 818 19 00381-1A Impa-Leu-Asp-Thr-Ser-Leu B,C 736 737 44 1140 20 00382-1A Bipa-Leu-Asp-Thr-Ser-Leu B,C 742 765 80 54 485 21 00383-1A Npa-Leu-Asp-Thr-Ser-Leu B,C 716 713 38,48 39 548 22 00384-1A Hbz-Leu-Asp-Thr-Ser-Leu B,C 750 770 77 51 401 23 00386-1A Pba-Leu-Asp-Thr-Ser-Leu B,C 818 817 94 88 (@50 &mgr;M) 2.8 24 00389-1A Ac-Leu-Asp-Thr-Ser-Leu B,C 710 711 61 29 205 25 00391-1A Tpc-Leu-Asp-Thr-Ser-Leu B,C 672 670 88 39 121 26 00392-1A Pha-Leu-Asp-Thr-Ser-Leu B,C 666 693 86 38 2128 27 00393-1A MeLeu-Leu-Asp-Thr-Ser-Leu B,C 905 928 86 54 256 28 00710-1A Tpa-Leu-Asp-Thr-Ser-Leu B,C 813 837 74 0.3 29 00711-1A Paa-Leu-Asp-Thr-Ser-Leu B,C 785 790 81 0.5 30 00781-1A Pba-Pro-Leu-Asp-Thr-Ser-Leu B,C 910 912 77 47 31 00784-1A Dpa-Leu-Asp-Thr-Ser-Leu B,C 737 765 87 11 32 00785-1A Dphb-Leu-Asp-Thr-Ser-Leu B,C 799 807 87 15 33 00786-1A Flc-Leu-Asp-Thr-Ser-Leu B,C 735 739 71 17 34 00787-1A Pca-Leu-Asp-Thr-Ser-Leu B,C 771 799 80 2.1 35 00788-1A Xnc-Leu-Asp-Thr-Ser-Leu B,C 751 757 84 3 36 00789-1A Pba-Leu-Asp-Thr-Ser-Leu B,C 813 818 81 4 37 00793-1A Tphp-Leu-Asp-Thr-Ser-Leu B,C 823 831 87 14 38 00795-1A 2-Anc-Leu-Asp-Thr-Ser-Leu B,C 747 756 63 5.4 39 00603-1A Pba-Leu-Ala-Thr-Ser-Leu B,C 771 775 84 100 (@250 &mgr;M) 28 40 00605-1A Ac-Leu-Phe-Thr-Ser-Leu B 623 623 86 29 299.7 41 00606-1A Ac-Leu-Glu-Thr-Ser-Leu B 605 605 85 98 60.7 42 00610-1A Pba-Leu-Tyr-Thr-Ser-Leu B,C 862 867 95 100 30 43 00612-1A Ac-Leu-Asp-Ser-Ser-Leu B 578 580 86 38 611.4 44 00613-1A Ac-Leu-Asp-Thr-Thr-Leu B 604 609 71 98 100 .7 45 00614-1A Ac-Leu-Asp-Thr-Ser-Phe B 625 625 80 86 143.9 46 00615-1A Ac-Lle-Asp-Thr-Ser-Leu B 591 593 79 17 18983 47 00712-1A Ac-Leu-Asp-Val-Ser-Leu B 589 611 93 71 48 00716-1A Ac-Leu-Asp-HyP-Ser-Leu B 599 626 79 106 49 00718-1A Ac-MeLeu-Asp-Thr-Ser-Leu B 587 629 65 150 50 00799-1A Cpc-Asp-Thr-Ser-Leu B,C 526 532 73 8.9 51 00649-1A Pba-Asp-Thr-Ser-Leu B,C 702 704 85 100 (@100 &mgr;M) 13.3 52 00800-1A Pca-Asp-Thr B,C 459 460 76 11 53 00650-1A Pba-Asp-Thr B,C 503 505 96 100 (@100 &mgr;M) 13.5 54 NH2-Leu-Asp-Thr-Ser-Leu 22 55 NH2-Trp-Arg-Gly-Leu-Asp-Thr-Ser-Leu- 76 Gly-Ser 56 NH2-His-Trp-Arg-Gly-Leu-Asp-Thr-Ser- 80 Leu-Gly-Ser-Val 57 NH2-Arg-Val-His-Trp-Arg-Gly-Leu-Asp- 90 Thr-Ser-Leu-Gly-Ser-Val-Gln 58 Ac-Leu-Asp-Thr-Ser-Leu 71 59 Ac-Arg-Gly-Leu-Asp-Thr-Ser-Leu-Gly 75 60 Ac-Trp-Arg-Gly-Leu-Asp-Thr-Ser-Leu-Gly- 71 Ser 61 Ac-His-Trp-Arg-Gly-Leu-Asp-Thr-Ser-Leu- 71 Gly-Ser-Val 62 Ac-Arg-Val-His-Trp-Arg-Gly-Leu-Asp-Thr- 88 Ser-Leu-Gly-Ser-Val-Gln

[0166] 5 TABLE 3 Ac = acetyl Anc = 2 Anthracenecarbonyl Bipa = 4-biphenylacetyl Bpc = benzofuranecarbonyl Chc = cyclohexanecarbonyl Cpa = 1-cyclopentylacetyl Cpc = cyclopentanecarbonyl DBU = diazobicyclo [5,40] undec-7-ene DMF = N,N-dimethylformanide Dpa = diphenylacety Dphb = 3,5-diphenylbenzoyl EDT = 1,2-ethanedithiol Fc = furanecarbonyl HBTU = 2(1-benzotriazolyl-1-yl-)-1,1,3,3-tetramethyluronium Hbz = 4-heptylbenzoyl Hyp = 4-hydroxyproline Idc = Indolecarbonyl Impa = 4-isobutyl-a-methylphenylacetyl Indc = Indanecarbonyl NMM = N-methylmorpholine Npa = a-naphthylacetyl Paa = 1-pyreneacetyl Pba = 1-pyrenebutyryl Pca = 1-pyrenecarbonyl Pha = phenylacetyl Phx = 6-phenylhexanoyl PYBOP = benzotriazolyl-N- oxytripyrrolidinophosphoniumhexafluorophosphate Rink Amide Am Resin = 4-[2′,4′-dimethoxyphenyl-Fmoc- aminomethyl]-phenoxylacetamido-norlecucylaminomethyl resin Tcc = trans-cinnamoyl TFA = trifluoroacetic acid Thc = Thienylcarbonyl Tpa = triphenylacetyl Tpc = tetramethylcyclopropylcarbonyl Tphp = triphenylpropionyl Xnc = xanthenecarbonyl

Example 3: Human MAdCAM Adhesion Assay

Design and Functional Analysis of a Human MAdCAM-1-IgG (huMAdCAM-1-Ig) Chimera

[0167] Construction of huMAdCAM-IgG Chimera

[0168] Human MAdCAM-1 clone 4 cDNA (FIG. 4, SEQ ID NO: 67), present in vector pCDNA3 (Invitrogen, San Diego, Calif.), was used as a template for PCR amplification of extracellular regions of human MAdCAM-1 to be fused with the constant region of a human IgGl. The human MAdCAM-1 clone 4 cDNA/pcDNA3 construct is also referred to as pcD3huMAd4 or pCDhuMAd4 (see Briskin et al., WO 96/24673, published Aug. 15, 1996, Example 1; see also Shyjan, A. M. et al., J. Immunol., 156: 2851-2857 (1996), the teachings of which are each incorporated herein by reference). In particular, primer HUMADIG4/2 (SEQ ID NO: 83), which contains the 5′ end of human MAdCAM-1 coding sequence (ATO codon, bold), was synthesized:

[0169] HUMADIG4/2 (SEQ ID NO: 83),

[0170] HindIIl

[0171] 5′-GGAAGCTTCCACCATGGATTTCGCACTGGCCC-3′

[0172] This 5′ primer was used in conjunction with a 3′ primer to amplify a region encoding the entire extracellular domain of human MAdCAM-1 (clone 4). The 3′ primer, designated HUMADIG3 (SEQ ID NO: 84), which contains a portion complementary to coding strand nucleotides 992-1010 of SEQ ID NO: 67, had the following sequence:

[0173] HUMADIG3 (SEQ ID NO: 84) 6      SpeI 5′-GGACTAGTGGTTTGGACGAGCCTGTTG-3′

[0174] The primers were designed with a 5′ HindIII site or 3′ SpeI sites as indicated. These primers were used to PCR amplify a MAdCAM fragment, using a PCR optimizer kit from Invitrogen (San Diego, Calif.). The PCR product was digested with the enzymes HindIII and SpeI to generate ends for cloning, and was purified by gel electrophoresis using the Glassmax DNA isolation system (Gibco, Bethesda, Md.).

[0175] A −1 kb fragment encompassing the CH1, H (hinge), CH2 and CH3 constant regions was excised by digestion with SpeI and EcoRI from a construct encoding a human immunoglobulin &ggr;1 heavy chain, having a constant region described by Reichmann, L. et al., Nature, 322: 323-327 (1988) and Takahashi, N. et al., Cell, 29: 671-679 (1982)), which further contained two mutations in the Fc region. The mutations in the Fc region of this Ig heavy chain construct (Leu235→Ala235 and Gly237→Ala237) were designed to reduce binding to human Fc&ggr; receptors, and were produced by oligonucleotide-directed mutagenesis. The antibody encoded by this construct was used as an isotype matched irrelevant control hereinbelow. The 1 kb SpeI-EcoRI fragment encoding the Fc-mutated IgG1 constant region was isolated by gel electrophoresis using the Glassmax DNA isolation system (Gibco, Bethesda Md.). This constant region fragment and the HindIII-SpeI fragment containing the entire extracellular domain were ligated in a three-way ligation to vector pEE12 (Stephens, P. L. and M. L. Cockett, Nucl. Acids Res., 17: 7110 (1989) and Bebbington, C. R. and C. C. G. Hentschel, 1987, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells, (Academic Press, N.Y.)), which had been digested with HindIII and EcoRI. Transformants of the bacterial strain DH10B were obtained. Colonies were grown and mini plasmid preps were analyzed by restriction mapping. A construct designated HuMAdIg21 (from clone 21), which encodes a fusion protein comprising the entire extracellular domain of MAdCAM-1 fused to the Fc-mutated IgG1 constant region, was sequenced across the entire MAdCAM-1 portion, confirming proper fusion of segments and the absence of PCR induced mutations.

[0176] For initial testing, the construct was transiently transfected onto monolayers of 5×107 COS cells in 1 ml of RPMI buffer (no serum) and 25 &mgr;g of plasmid using electroporation with a Biorad Gene Pulser under standard conditions (960 &mgr;F, 250 V). 72-96 hours after transfection, supernatants were harvested, passaged through 0. 45 &mgr; filters and stored at 40° C. in the presence of 0.05% sodium azide. Production of chimera was confirmed by a sandwich ELISA, using an anti-human IgG1 antibody as capture antibody and the same antibody, which was conjugated to alkaline phosphatase as second antibody for detection. Irrelevant control antibody (having an identical constant region) was used as a standard. The chimera was also analyzed by Western blotting using an anti-human MAdCAM-1 monoclonal antibody, and was found to run at approximately 200 kd, consistent with the size of a homodimer.

[0177] Soluble Human MAdCAM-Ig Chimeras Specifically Bind &agr;4&bgr;7 Positive Cells

[0178] Supernatants from two different transfections were assayed for their ability to stain the T cell line HUT 78, which was previously shown to bind MAdCAM-1 only in the presence of Mn++. Accordingly, each solution used in this assay contained 2 mM Mn++. HUT 78 cells (a human T cell lymphoma line; American Type Culture Collection, Accession No. ATCC TIB 161) are &agr;4&bgr;7-bearing cells. To test the binding specificity of the chimeras, HUT 78 cells were preincubated with either media alone (RPMI 1640 with 2% FCS) or media and 10 &mgr;g/ml of the anti-&bgr;7 antibody FIB 504. Approximately 100,000 cells were incubated on ice for 15 minutes and then washed with HBSS plus 2% FCS/2 mM Ca++/2 mM Mn++. Cells were then incubated for 20 minutes on ice with media once again, or with supernatants from one of two independent transfections with the construct encoding the chimera comprising the entire extracellular domain of MAdCAM-1 (HuMAdIg21). After washing, cells were then incubated with an anti-human IgG antibody conjugated with phycoerythrin and staining above background was assessed by flow cytometry (FACScan). Only cells incubated with the chimera supernatants stained above background, while preincubation with the &bgr;7 MAb reduced this staining to background levels, indicating a specific interaction of the chimera with the &agr;4&bgr;7 integrin.

[0179] Permanent NSO cell lines secreting human MAdCAM-Ig chimera were selected after transfection by electroporation, by growth in a glutamine free media as previously described (Cockett, M. L., et al., Bio/Technology, 8: 662-667 (1990)). Cloned lines were adapted to growth in spinner culture. Supernatants from three of these cloned lines (samples B-D), and a partially purified chimera (Clone 21, purified by binding to protein A, sample A) were tested for their ability to support adhesion of the B cell line RPMI 8866. Briefly, NEN maxisorb plates were incubated with 100 &mgr;L/well of Protein A at 20 &mgr;g/ml in carbonate buffer, pH 9.5 overnight at 4° C. Plates were then washed 2X with RPMI 1640 media (no serum). 100 &mgr;l of chimera (or serial dilutions in RPMI) were bound to the wells at 37° C. for 2 hours and then washed once. Wells were then blocked with FCS for 1 hour at 37° C., washed once, and then preincubated with tissue culture supernatants containing either an anti-human VCAM-1 MAbs (2G7) as a control or the anti-human MAdCAM-1 MAb 10G3 (Briskin et al., WO 96/24673, Example 2). 2G7 and 10G3 MAbs were removed before addition of cells. RPMI 8866 cells were fluorescently labeled by preincubation with BCECF-AM stain (BCECF-AM; 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyflourescein, acetoxymethyl ester; Molecular Probes), 100 &mgr;l of cells were added to each well (to a final concentration of 105 cell/well), and incubated on a rotary shaker for 30 minutes at room temperature. Binding of RPMI 8866 cells to immobilized chimeras was assessed by reading flourescence values using a Fluorescence Concentration Analyzer (IDEXX). Specific binding was demonstrated as only the anti-human MAdCAM-1 MAb could block binding of cells to MAdCAM-Ig chimera (Table 3A). 7 TABLE 3A Anti HuMAd Sample MAbs Neat 1:2 1:4 1:8 1:16 1:32 A − 195527 195527 195527 18560 4254 3558 + 3860 3092 1746 2200 482 564 B − 195527 195527 195527 195528 52056 2932 + 6526 3626 3274 2978 1648 1518 C − 195527 195527 35548 9570 21782 1926 + 4566 4094 3922 3492 2436 2566 D − 195527 30840 46852 16270 5474 2656 + 7350 6794 6020 4510 6548 5122 The &agr;4&bgr;7 Positive B Cell Line RPMI 8866 Specifically Binds Soluble Human MAdCAM Ig Chimera. The Human MAdCAM-1 Ig Chimera Clone 21 that was partially purified over protein A (Sample A) or tissue culture supernatants from different NSO clones (Samples B-D) were immobilized on 96 well plates with protein A, and either pre-incubated with an anti-VCAM-1 MAb 2G7 (designated “−” under MAbs) as a negative control or with the anti-human MAdCAM MAb 10G3 (“+”). #Purified chimera or supernatants (used either undiluted (“neat”) or at serial 1:2 dilutions) was bound to wells via Protein A, and incubated with fluorescently-labeled RPMI 8866 cells on a rotary shaker for 30 minutes at room temperature. After washing with an automated plate washer (EL 404 Microplate Autowasher, BIO-TEK Instruments), bound cells were counted with an automated plate reader (IDEXX). Raw numbers are thus a reflection of numbers of cells bound.

[0180] Production and Purification of huMAdCAM-1-Ig Chimera

[0181] NS0 clone 21-9C10 producing huMAdCAM-1-Ig from HuMAdIG21 was prepared as described above. The clone was grown in glutamine free DMEM, 10% ultra low bovine Ig FCS, 1X GS supplement (Cibco Inc.), (a mixture of non-essential amino acids and nucleosides for addition to glutamine-free basal media), 1 mM pyruvate, 50 units/ml penicillin G and 50 &mgr;g/ml streptomycin sulfate. huMAdCAM-1-Ig producing clones were adapted to grow in 1.0 liter spinner flasks at 37° C., 5% CO2, 95s air and a constant stir rate of 60-70 rpm. Cultures were seeded at a cell density of 1 to 2×105 cells/ml in 250 mls of the above media and allowed to grow for 48-72 hours before bringing the final volume of the culture to 550 to 600 mls. Spinner flasks were harvested at day 10-11 and centrifuged at 6,000 rpm, 4° C., 25 minutes. The resulting culture supernatants were then 0.2 &mgr; filtered and stored at 4° C.

[0182] huMAdCAM-1-Ig culture supernatants were passed over a 20 ml bed volume Protein A column, at equilbrated in PBS, pH 7.2, at 1.5 mls/min flow rate. The column was then washed at 5.0 mls/min with PBS, pH 7.2 at 1.5 mls/min flow rate. The column was then washed at 5.0 mls/min with PBS pH 7.2 and eluted with 0.1 M citrate, pH 3.5 at a flow rate of 5.0 mls/min. 5.0 ml fractions were collected and neutralized immediately with 250 &mgr;l/fraction 1.5 M sodium carbonate, pH 11.5. The Protein A column was then equilibrated with PBS, pH 7.2.

[0183] The resulting fractions from Protein A purification were analyzed by an anti-human Ig(Fc) (Jackson Research Labs.) ELISA. Peak fractions containing huMAdCAM-1-Ig were concentrated by centrifugacion at 6,000 rpm, 4° C. with Centricon 30 concentrators (30,000 mw cutoff).

[0184] Final product was quantitated by BioRad Protein Assay, analyzed for biological activity in the huMAdCAM-1-Ig/RPMI 8866 Adhesion Assay (see below) and for purity with 4-20% SDS-PAGE, reduced and nonreduced, by colloidal coomassie blue staining.

[0185] Adhesion Assay Protocol

[0186] RPMI 8866 cells, a B cell lymphoma expressing &agr;4&bgr;7 (a gift from D. Erle; Erle, D. J. et al., J. Immunol., 153:517-528 (1994); Shyjan et al., Journal of Immunology, 1996:2851 (1996) were fluorescently labeled (labeling protocol provided below) using BCECF (Molecular probes #B-1170). Cells were resuspended in an assay buffer consisting of Hanks Balanced Salt Solution (HBSS) supplemented with fetal calf serum (FCS) at a final concentration of 2%, HEPES, pH 7.3 at 25 mM. All reagents in this buffer were supplied from Gibco/BRL.

[0187] All assays were performed in 96 well strip well plates from Costar (E.I.A./R.I.A. Strip plate-8 Cat. #2581). Protein A purified human MAdCAM-1-Ig chimera was suspended in carbonate buffer (0.2 M NaHCO3 and 0.8 M Na2CO3, pH 9.5) at a concentration of 200 ng/ml and 50 &mgr;l was added to each well for a plating concentration of 10 ng/well. The plates were incubated at 37° C. for one hour (or at 4° C. overnight) and washed once in the automatic plate washer (50 mM Tris HCl, 0.14 M NaCl and 2.0 mM MnCl2 at pH 7.2) (Bio-Tek EL 404 microplate autowasher). Wells were then blocked with PBS (phosphate buffered saline) and 10% FCS (100 &mgr;l/well) for one hour at 37° C. followed by the same wash step as described.

[0188] Cells were labeled with BCECF-AM (Molecular Probes, Cat. No. B-1170) at a concentration of 2 &mgr;l/ml to a cell suspension of 4×106 cells/ml in PBS. The cells were incubated in a 37° C. water bath for 30 minutes. Labeled cells were centrifuged at 700 RPM for 10 minutes, resuspended in assay buffer and the centrifugation step was repeated. The cells were resuspended at a final concentration of 2.5×106 cells/ml.

[0189] For assays, compounds were diluted as follows in a 96 well polypropylene plate. First, a stock solution of each compound was prepared by dissolving compound in 100% DMSO at 100X final concentration to be tested. 10 &mgr;l of the stock was added to 90 &mgr;l water for the first test dilution. 50 &mgr;l of the first test dilution was added to 50 &mgr;l of 10% DMSO in water for the second test dilution. 50 &mgr;l of the second test dilution was added to 50 &mgr;l of 10% DMSO in water for the third test dilution. 50 &mgr;of the third test dilution was added to 50 &mgr;l of 10% DMSO in water for the fourth test dilution, and so on, until six test dilutions were prepared.

[0190] A stock solution of SmM positive control peptide (L783-1A) was made in 100% DMSO, and subsequently diluted in water 1:10 (10 &mgr;l of 5 mM stock plus 90 &mgr;l of water) to yield a concentration of 0.5mM. This dilution was then further diluted 1:2 with 10% DMSO (50 &mgr;l of the compound and 50 &mgr;l of 10% DMSO) for a concentration of 250 &mgr;M. These 1:2 dilutions were continued serially until six dilutions were achieved. These dilutions represent 10X stocks as shown in the assay set up below:

[0191] The assay was set up by adding materials to huMAdCAM-1-Ig-coated plates as follows:

[0192] 1) 130 &mgr;l/well of assay buffer

[0193] 2) 20 &mgr;l/well of diluted compounds or 10% DMSO (control) or diluted positive control peptide.

[0194] 3) 50 &mgr;l of ECECF-AM-labeled RPMI 8866 cells

[0195] In this example, the final concentration of cells was 1.25×106 cells/well and the final concentration of compound ranged in a six point assay from 50 &mgr;m to 1.06 &mgr;m.

[0196] The plates were wrapped in foil and incubated for 30 minutes at room temperature on a rotary shaker at 45 RPM.

[0197] Plates were washed twice (50 mM Tris HCl, 0.14 mM NaCl and 2.0 mM MnCl2 at pH 7.2) on the microplate autowasher plate washer set as follows:

[0198] Wash Volume=500 ml

[0199] Wash Cycle=2X

[0200] Soak Time=0

[0201] Wash Depth=80

[0202] Aspirate After Last Wash

[0203] Shake Time=0

[0204] Plates were read in an IDEX fluoresecent plate reader set to excite at 485 nm and read at 535 nm.

[0205] Data was collected in microsoft excel in a format for automatic IC 50 determinations. The IC 50 is the fluorescence level that is 50% of maximum binding. Results are shown in Tables 4-7.

[0206] In Table 4, Example No. 1478 is represented by SEQ ID NO: 71; Example No. 783 is represented by SEQ ID NO: 72; Example No. 1492 is represented by SEQ ID NO: 73; Example No. 1487 is represented by SEQ ID NO: 74; Example No. 1490 is represented by SEQ ID NO: 75; Example No. 1275 is re prese nted by SEQ ID NO: 76; Example No. 1274 is represented by SEQ ID NO: 77; Example No. 1282 is represented by SEQ ID NO: 78; Example No. 1481 is represented by SEQ ID NO: 79; Example No. 1482 is represented by SEQ ID NO: 80; Example No. 1486 is represented by SEQ ID NO: 81; Example No. 1498 is represented by SEQ ID NO: 82. 8 TABLE 4 R1-Leu-Asp-Thr-Ser-Leu Method Mass Mass hMAdCAM L # R1 Prep Calc. Found IC50 1478 3 C 647 658 11.2  783 4 C 673 701 10 1492 5 C 687 715 2.1 1487 6 C 686 690 3 1490 7 C 720 724 1.7 1275 8 C 698 726 3.5 1276 9 C 698 703 2.7 1282 10 C 699 706 4 1481 11 C 698 705 3.2 1482 12 C 698 700 2.2 1486 13 C 698 705 1.4 1491 14 C 649 658 3.4

[0207] 9 TABLE 5 R1-Leu-Asp-Thr Method Mass Mass hMAdCAM L # R1 Prep Calc. Found IC50 1496 15 C 448 503 8.2 1495 16 C 499 501 3.1 2277 17 C 584 587 1.7 2278 18 C 570 575 0.4 2273 19 C 618 620 1.2 2271 20 C 607 601 0.9 2261 21 C 632 633 0.86 2263 22 C 621 622 1.3 2262 23 C 654 679 1.5 2279 24 C 632 636 1

[0208] 10 TABLE 6 R1-Leu-Asp-Thr Method Mass Mass hMAdCAM Example R1 Prep Calc. Found IC50 1 25 C 550 550 0.58 2 26 C 518 518 0.73 3 27 C 484 484 1.4 4 28 C 506 506 1.1 5 29 C 493 493 1.5 6 30 C 535 534 2.6 7 31 C 518 518 2.8 8 32 C 486 486 2 9 33 C 499 499 1.1

[0209] 11 TABLE 7 R1-Leu-Asp-Thr-R2 Method Mass Mass hMAdCAM Example R1-Leu-Asp-Thr-R2 Prep Calc. Found IC50 1 34 F 617 617 0.78 2 35 F 583 583 0.94 3 36 F 599 599 0.97 4 37 F 609 609 0.64 5 38 F 605 605 0.81 6 39 F 655 655 0.97 7 40 F 541 541 3.8 8 41 F 611 611 1.3 9 42 F 545 545 1.5 10 43 F 606 617 1.5

EQUIVALENTS

[0210] Those skilled in the art will be able to recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A compound represented by the following structural formula:

R1—X—Y′—Z′R2

wherein:

Y′ is a pentapeptide having the sequence Leu-Asp-Thr-Ser-Leu (SEQ ID NO: 1), Xaa-Asp-Thr-Ser-Leu (SEQ ID NO: 85), Leu-Xaa-Thr-Ser-Leu (SEQ ID NO: 86), Leu-Asp-Xaa-Ser-Leu (SEQ ID NO: 87), Leu-Asp-Thr-Xaa-Leu (SEQ ID NO: 88), or Leu-Asp-Thr-Ser-Xaa (SEQ ID NO: 89);

Xaa is a naturally-occurring amino acid;

X and Z are independently chosen from the group consisting of a covalent bond, an amino acid or a peptide, wherein each amino acid in X and Z is independently selected from the group of naturally occurring amino acids;

R1 is R3—CO—;

R2 is —NR4R5;

R3 is selected from the group consisting of a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group;

R4 and R5 are each independently selected from the group consisting of hydrogen, a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, wherein:

1) R4 and R5 are not both —H; and

2) taken together, R4 and R5 can form a heterocyclic ring;

taken together, X, Y′ and Z form a peptide containing no more than about fifteen amino acids; and

wherein optionally the peptide formed from X, Y′ and Z is cyclized.

2. The compound of claim 1 wherein R3 is selected from the group consisting of triphenylmethyl, diphenylmethyl, 3,5-diphenylphenyl, 2-furanyl, 3-furanyl, 9-xanthenemethyl, 2,2,2-triphenylethyl, 2-anthracene, methyl, cyclopentyl, 2-indolyl, 2-indanyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, cyclohexyl, 5-phenylpentyl, 4-isobutyl-&agr;-methylphenylmethyl, 4-biphenylmethyl, &agr;-naphthylmethyl, 4-heptylphenyl, phenylmethyl, trans 2-phenylethenyl and 2,2,3,3-tetramethylcyclopropyl.

3. The compound of claim 2 wherein R4 and R5 are each independently selected from the group consisting of —H, 2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, -CH2-2-thienyl, -CH2-3-thienyl, —CH2-2-furanyl, —CH2-3-furanyl, 3,4-dimethoxybenzyl, and isopentyl.

4. The compound of claim 3 wherein Y, is Leu-Asp-Thr-Ser-Leu (SEQ ID NO: 1).

5. The compound of claim 1 wherein:

R3 is selected from the group consisting of diphenylmethyl, triphenylmethyl, trans 2-phenyl-ethylenyl, 2-phenyl-ethynyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl and 3-benzothienyl;

R4 is selected from the group consisting of 2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, —CH2-2-thienyl, —CH2-3-thienyl, —CH2-2-furanyl, —CH2-3-furanyl; and

R5 is —H.

6. The compound of claim 5 wherein X and Z are each a covalent bond.

7. The compound of claim 1 wherein the peptide formed from X, Y, and Z is cyclized.

8. A compound represented by the following structural formula:

R1—Y′—R2

wherein:

Y′ is a pentapeptide having the sequence Leu-Asp-Thr-Ser-Leu (SEQ ID NO: 1), Xaa-Asp-Thr-Ser-Leu (SEQ ID NO:85), Leu-Xaa-Thr-Ser-Leu (SEQ ID NO: 86), Leu-Asp-Xaa-Ser-Leu (SEQ ID NO: 87), Leu-Asp-Thr-Xaa-Leu (SEQ ID NO: 88), or Leu-Asp-Thr-Ser-Xaa (SEQ ID NO: 89);

Xaa is a naturally-occurring amino acid;

R1 is R3—CO—;

R2 i s —NR4R5;

R3 is selected from the group consisting of a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group; and

R4 and R5 are each independently selected from the group consisting of hydrogen, a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, wherein:

1) R4 and R5 are not both —H; and

2) taken together, R4 and R5 can form a heterocyclic ring; and

wherein optionally Y is cyclized.

9. The compound of claim 8 wherein Y′ has the sequence Leu-Asp-Thr-Ser-Leu (SEQ ID NO: 1).

10. The compound of claim 9 wherein R3 is selected from the group consisting of monocyclic and bicyclic nitrogen-containing heteroaromatic groups, vinyl groups substituted with substituted and unsubstituted aryl and heteroaryl groups, polycarbocyclic aromatic hydrocarbons and oxygen-containing polycyclic aromatic hydrocarbons.

11. The compound of claim 9 wherein R3 is selected from the group consisting of a quinolinyl group, an isoquinolinyl group, an indolyl group, a quinoxalinyl group, a cinnolinyl group, a pyrazinyl group, a styryl group, a stilbyl group, (3-pyridyl)—CH═CH—, a naphthyl group, an anthracyl group, a xanthanyl group, a benzopyranone group and a benzofuranyl group.

12. A compound represented by the following structural formula:

R1—X—Y′—Z—R2

wherein:

Y′ is a tripeptide [AA]1-[AA]2-[AA]3 having the sequence Leu-Asp-Thr;

X and Z are independently chosen from the group consisting of a covalent bond, an amino acid or a peptide, wherein each amino acid in X and Z is independently selected from the group of naturally occurring amino acids;

R1 is R3—CO—;

R2 is —NR4R5;

R3 is selected from the group consisting of a lower alkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and

R4 and R5 are each independently selected from the group consisting of hydrogen, a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, wherein:

1) R4 and R5 are not both —H; and

2) taken together, R4 and R5 can form a heterocyclic ring; and

taken together, X, Y′ and Z form a peptide containing no more than about fifteen amino acids; and

wherein optionally the peptide formed from X, Y′ and Z is cyclized.

13. The compound of claim 12 wherein R3 is selected from the group consisting of triphenylmethyl, diphenylmethyl, 3,5-diphenylphenyl, 2-furanyl, 3-furanyl, 9-xanthenemethyl, 2,2,2-triphenylethyl, 2-anthracene, methyl, cyclopentyl, 2-indolyl, 2-indanyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, cyclohexyl, 5-phenylpentyl, 4-isobutyl-&agr;-methylphenylmethyl, 4-biphenylmethyl, &agr;-naphthylmethyl, 4-hepzylphenyl, phenylmethyl, trans 2-phenylethenyl and 2,2,3,3-tetramethylcyclopropyl.

14. The compound of claim 13 wherein R4 and R5 are each independently selected from the group consisting of —H, 2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, -CH2-2-thienyl, -CH2-3-thienyl, —CH2-2-furanyl, —CH2-3-furanyl, 3,4-dimethoxybenzyl, and isopenzyl.

15. The compound of claim 12 wherein:

R3 is selected from the group consisting of diphenylmethyl, triphenylmethyl, trans 2-phenyl-ethylenyl, 2-phenyl-ethynyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl and 3-benzothienyl;

R4 is selected from the group consisting of 2-hydroxyethyl benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, —CH2-2-thienyl, —CH2-3-thienyl, —CH2-2-furanyl, —CH2-3-furanyl; and

R5 is —H.

16. The compound of claim 12 wherein the peptide formed from X, Y′ and Z is cyclized.

17. A compound represented by the following structural formula:

R1—Y′—R2

wherein:

Y′ is a tripeptide [AA]1-[AA]2-[AA]3 having the sequence Leu-Asp-Thr;

R1 is R3—CO—;

R2 is —NR4R5;

R3 is selected from the group consisting of a lower alkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and

R4 and R5 are each independently selected from the group consisting of hydrogen, a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, wherein:

1) R4 and R5 are not both —H; and

2) taken together, R4 and R5 can form a heterocyclic ring.

18. The compound of claim 17 wherein R3 is selected from the group consisting of phenyl, substituted phenyl, thienyl, substituted thienyl, indolyl, substituted indolyl, pyrimidyl, substituted pyrimidyl, benzofuranyl, substituted benzofuranyl, quinolinyl, substituted quinolinyl, isoquinolinyl, substituted isoquinolinyl, benzopyranone groups, substituted benzopyranone groups and 3-isoquinolinyl-CO—NH—(CH2)x—, wherein x is an integer from 1-4.

19. The compound of claim 18 wherein:

R3 is 3-isoquinolinyl or 2-benzofuranyl;

R4 is —H; and

R5 is benzyl, substituted benzyl, phenethyl, substituted phenethyl, phenpropyl, substituted phenpropyl, heteroaryl-CH2—, substituted heteroaryl-CH2—, lower alkyl, substituted lower alkyl, cycloalkyl and a group represented by one of the following structural formulas:

44

20. The compound of claim 18 wherein:

R3 is 3-isoquinolinyl or 2-benzofuranyl; and

R4 and R5, taken together, form a heterocyclic ring selected from the group consisting of pyrrolidinyl, substituted pyrrolidinyl, indoline, isomers of indoline, substituted indoline, substituted isomers of indoline, tetrahydroisoquinoline, substituted tetrahydroisoquinoline, tetrahydroquinoline, substituted tetrahydroquinoline, piperidone, substituted piperidone, piperidine, substituted piperidines, tetrahydro-oxazines and substituted tetrahydro-oxazines.

21. The compound of claim 17 wherein R1 is represented by the following structural formula:

45

wherein:

A is selected from the group consisting ofi an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroarylgroup; and

n and m are each independently zero or one.

22. A compound represented by the following structural formula:

R1—X—Y′—Z—R2

wherein:

Y′ is a dipeptide [AA]1-[AA]2 having the sequence Asp-Thr;

X and Z are independently chosen from the group consisting of a covalent bond, an amino acid or a peptide, wherein each amino acid in X and Z is independently selected from the group of naturally occurring amino acids;

R1 is R3—CO—;

R2 is —NR4R5;

R3 is selected from the group consisting of a lower alkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and

R4 and R5 are each independently selected from the group consisting of hydrogen, a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, wherein:

1) R4 and R5 are not both —H; and

2) taken together, R4 and R5 can form a heterocyclic ring; and

taken together, X, Y′ and Z form a pentide containing no more than about fifteen amino acids; and

wherein optionally the peptide formed from X, Y′ and Z is cyclized with the proviso that, if the peptide formed from X, Y′ and Z is cyclized, the nitrogen at the N-terminus of Y′ is not bonded to a glycine or a sarcosine.

23. The compound of claim 22 wherein R3 is selected from the group consisting of triphenylmethyl, diphenylmethyl, 3,5-diphenylphenyl, 2furanyl, 3-furanyl, 9-xanthenemethyl, 2,2,2-triphenylethyl, 2-anthracene, methyl, cyclopentyl, 2-indolyl, 2-indanyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, cyclohexyl, 5-phenylpentyl, 4-isobutyl-&agr;-methylphenylmethyl, 4-biphenylmethyl, a-naphthylmethyl, 4-heptylphenyl, phenylmethyl, trans 2-phenylethenyl and 2,2,3,3-tetramethylcyclopropyl.

24. The compound of claim 23 wherein R4 and R5 are each independently selected from the group consisting of —H, 2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, -CH2-2-thienyl, -CH2-3-thienyl, —CH2-2-furanyl, —CH2-3-furanyl, 3,4-dimethoxybenzyl, and isopentyl.

25. The compound of claim 22 wherein:

R3 is selected from the group consisting of R3 is selected from the group consisting of diphenylmethyl, triphenylmethyl, trans 2-phenyl-ethylenyl, 2-phenyl-ethynyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl and 3-benzothienyl;

R4 is selected from the group consisting of 2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, —CH2-2-thienyl, —CH2-3-thienyl, —CH2-2-furanyl, —CH2-3-furanyl; and

R5 is —H.

26. The compound of claim 25 wherein X and Z are each a covalent bond.

27. The compound of claim 22 wherein the peptide formed from X, Y, and Z is cyclized.

28. A method of treating an individual suffering from a disease associated with leukocyte infiltration of tissues expressing the molecule MAdCAM-1, comprising administering a therapeutically effective amount of an inhibitor represented by the following structural formula:

R1—X—Y—Z—R2

wherein:

Y is a pentapeptide [AA]1-[AA]2-[AA]3 -[AA]4-[AA]5 wherein:

[AA]1 is selected from the group consisting of leucine, cysteine, aspartic acid, glutamic acid, isoleucine, alanine, valine, phenylalanine, glycine, N-methylleucine, serine, threonine, ornithine and lysine;

[AA]2 is selected from the group consisting of aspartic acid, glutamic acid, phenylalanine and tyrosine;

[AA]3 is selected from the group consisting of threonine, serine, valine, proline and 4-hydroxyproline;

[AA]4 is selected from the group consisting of serine, cysteine, aspartic acid, glutamic acid, proline, 4-hydroxyproline, threonine, valine, isoleucine, alanine, glycine, ornithine and lysine; and

[AA]5 is selected from the group consisting of leucine, isoleucine, N-methylleucine, threonine, ornithine, serine, valine, alanine, glycine, phenylalanine, cysteine, aspartic acid, glutamic acid and lysine;

X and Z are independently chosen from the group consisting of a covalent bond, an amino acid or a peptide, wherein each amino acid in X and Z is independently selected from the group of naturally occurring amino acids;

R1 is R3—CO—;

R2 is —NR4R5;

R3 is selected from the group consisting of a lower alkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and

R4 and R5 are each independently selected from the group consisting of hydrogen, a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, wherein:

1) R4 and R5 are not both —H; and

2) taken together, R4 and R5 can form a heterocyclic ring; and

taken together, X, Y and Z form a peptide containing no more than about fifteen amino acids; and

wherein:

optionally the peptide formed by X, Y and Z is cyclized; and

an arginine or an arginine derivative is not bonded to the nitrogen at the N-terminus of Y.

29. The method of claim 28 wherein Y is a pentapeptide having the sequence Leu-Asp-Thr-Ser-Leu (SEQ ID NO: 1), Xaa-Asp-Thr-Ser-Leu (SEQ ID NO: 85), Leu-Xaa-Thr-Ser-Leu (SEQ ID NO: 86), Leu-Asp-Xaa-Ser-Leu (SEQ ID NO: 87), Leu-AsD-Thr-Xaa-Leu (SEQ ID NO: 88), or Leu-Asp-Thr-Ser-Xaa (SEQ ID NO: 89), wherein Xaa is a naturally-occurring amino acid.

30. The method of claim 29 wherein R3 is selected from the group consisting of triphenylmethyl, diphenylmethyl, 3,5-diphenylphenyl, 2-furanyl, 3-furanyl, 9-xanthenemethyl, 2,2,2-triphenylethyl, 2-anthracene, methyl, cyclopentyl, 2-indolyl, 2-indanyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, cyclohexyl, 5-phenylpentyl, 4-isobutyl-&agr;-methylphenylmethyl, 4-biphenylmethyl, a-naphthylmethyl, 4-heptylphenyl, phenylmethyl, trans 2-phenylethenyl and 2,2,3,3-tetramethylcyclopropyl.

31. The method of claim 30 wherein R4 and R5 are each independently selected from the group consisting of —H, 2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, —CH2-2-thienyl, —CH2-3-thienyl, —CH2-2-furanyl, —CH2-3-furanyl, 3,4-dimethoxybenzyl, and isopentyl.

32. The method of claim 28 wherein:

[AA]1 is selected from the group consisting of leucine, valine, isoleucine, alanine, glycine, phenylalanine and N-methylleucine;

[AA]2 is selected from the group consisting or aspartic acid, glutamic acid, phenylalanine and tyrosine;

[AA]3 is selected from the group consisting of threonine, serine, valine, proline and 4-hydroxyproline;

[AA]4 is selected from the group consisting of serine, cysteine and threonine; and

[AA]5 is selected from the group consisting of alanine, valine, leucine, isoleucine, alanine, glycine, phenylalanine and N-methylleucine.

33. The method of claim 32 wherein Y is Leu-Asp-Thr-Ser-Leu (SEQ ID NO: 1).

34. The method of claim 28 wherein:

R3 is selected from the group consisting of diphenylmethyl, triphenylmethyl, trans 2-phenyl-ethylenyl, 2-phenyl-ethynyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl and 3-benzothienyl;

R4 is selected from the group consisting of 2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzoth4enyl, 3-benzothienyl, —CH2-2-thienyl, —CH2-3-thienyl, —CH2-2-furanyl, —CH2-3-furanyl;

and R5 is —H.

35. The method of claim 28 wherein the peptide formed from X, Y and Z is cyclized.

36. A method of treating an individual suffering from a disease associated with leukocyte infiltration of tissues expressing the molecule MAdCAM-1, comprising administering a therapeutically effective amount of an inhibitor represented by the following structural formula:

R1—Y′—R2

wherein:

Y′ is a pentapeptide having the sequence Leu-Asp-Thr-Ser-Leu (SEQ ID NO: 1), Xaa-Asp-Thr-Ser-Leu (SEQ ID NO: 85), Leu-Xaa-Thr-Ser-Leu (SEQ ID NO: 86), Leu-Asp-Xaa-Ser-Leu (SEQ ID NO: 87), Leu-Asp-Thr-Xaa-Leu (SEQ ID NO: 88), or Leu-Asp-Thr-Ser-Xaa (SEQ ID NO: 89);

Xaa s a naturally-occurring amino acid;

R1 is R3—CO—;

R2 is —NR4R5;

R3 is selected from the group consisting of a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group; and

R4 and R5 are each independently selected from the group consisting of hydrogen, a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, wherein:

1) R4 and R5 are not both —H; and

2) taken together, R4 and R5 can form a heterocyclic ring; and

wherein optionally Y is cyclized.

37. The method of claim 36 wherein Y′ has the sequence Leu-Asp-Thr-Ser-Leu (SEQ ID NO: 1).

38. The method of claim 37 wherein R3 is selected from the group consisting of a monocyclic and bicyclic nitrogen-containing heteroaromatic groups, vinyl groups substituted with substituted and unsubstituted aryl and heteroaryl groups, polycarbocyclic aromatic hydrocarbons and oxygen-containing polycyclic aromatic hydrocarbons.

39. The method of claim 38 wherein R3 is selected from the group consisting of a quinolinyl group, an isoquinolinyl group, an indolyl group, a quinoxalinyl group, a cinnolinyl group, a pyrazinyl group, a styryl group, a stilbyl group, (3-pyridyl)—CH═CH—, a naphthyl group, an anthracyl group, a xanthanyl group, a benzopyranone group and a benzoFuranyl group.

40. The method of claim 28 wherein the disease is selected from the group consisting of inflammatory bowel disease and insulin-dependent diabetes mellitus.

41. A method of treating an individual suffering from a disease associated with leukocyte infiltration of tissues expressing the molecule MAdCAM-1, comprising administering a therapeutically effective amount of an inhibitor represented by the following structural formula:

R1—X—Y′—Z—R2

wherein:

Y′ is a tripeptide [AA]1-[AA]2-[AA]3 having the sequence Leu-Asp-Thr;

X and Z are independently chosen from the group consisting of a covalent bond, an amino acid or a peptide, wherein each amino acid in X and Z is independently selected from the group of naturally occurring amino acids;

R1 is R3—CO—;

R is —NRR;

R3is selected from the group consisting of a lower alkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and

R4 and R5 are each independently selected from the group consisting of hydrogen, a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, wherein:

1) R4 and R5 are not both —H; and

2) taken together, R4 and R5 can form a heterocyclic ring; and

taken together, X, Y′ and Z form a peptide containing no more than about fifteen amino acids; and

wherein optionally the peptide formed from X, Y′ and Z is cyclized.

42. The method of claim 41 wherein R3 is selected from the group consisting of triphenylmethyl, diphenylmethyl, 3,5-diphenylphenyl, 2-furanyl, 3-furanyl, 9-xanthenemethyl, 2,2,2-triphenylethyl, 2-anthracene, methyl, cyclopentyl, 2-indolyl, 2-indanyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, cyclohexyl, 5-phenylpentyl, 4-isobutyl-&agr;-methylphenylmethyl, 4-biphenylmethyl, &agr;-naphthylmethyl, 4-heptylphenyl, phenylmethyl, trans 2-phenylethenyl and 2,2,3,3-tetramethylcyclopropyl.

43. The method of claim 42 wherein R4 and R5 are each independently selected from the group consisting of —H, 2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, —CH2-2-thienyl, —CH2-3-thienyl, —CH2-2-furanyl, —CH2-3-furanyl, 3,4-dimethoxybenzyl, and isopentyl.

44. The method of claim 41 wherein:

R3 is selected from the group consisting of diphenylmethyl, triphenylmethyl, trans 2-phenyl-ethylenyl, 2-phenyl-ethynyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl and 3-benzothienyl;

R4 is selected from the group consisting of 2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, —CH2-27thienyl, —CH2-3-thienyl, —CH2-2-furanyl, —CH2-3-furanyl;

and R5 is —H.

45. The method of claim 42 wherein the peptide formed from X, Y′ and Z is cyclized.

46. The method of claim 41 wherein the disease is selected from the group consisting of inflammatory bowel disease and insulin-dependent diabetes mellitus.

47. A method of treating an individual suffering from a disease associated with leukocyte infiltration of tissues expressing the molecule MAdCAM-1, comprising administering a therapeutically effective amount of an inhibitor represented by the following structural formula:

R1—Y′—R2

wherein:

Y′ is a tripeptide [AA]1-[AA]2-[AA]3 having the sequence Leu-Asp-Thr;

R1 is R3—CO—;

R2 is —NR4R5;

R3 is selected from the group consisting of a lower alkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and

R4 and R5 are each independently selected from the group consisting of hydrogen, a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, wherein:

1) R4 and R5 are not both —H; and

2) taken together, R4 and R5 can form a heterocyclic ring.

48. The method of claim 47 wherein R3 is selected from the group consisting of phenyl, substituted phenyl, thienyl, substituted thienyl, indolyl, substituted indolyl, pyrimidyl, substituted pyrimidyl, benzofuranyl, substituted benzofuranyl, quinolinyl, substituted quinolinyl, isoquinolinyl, substituted isoquinolinyl, benzopyranone groups, substituted benzopyranone groups, and 3-isoquinolinyl—CO—NH—(CH2)x, wherein x is an integer from 1-4.

49. The method of claim 48 wherein:

R3 is 3-isoquinolinyl or 2-benzofuranyl;

R4 is —H; and

R5 is benzyl, substituted benzyl, phenethyl, substituted phenethyl, phenpropyl, substituted phenpropyl, heteroaryl-CH2—, substituted heteroaryl-CH2—, lower alkyl, substituted lower alkyl, cycloalkyl, substituted cycloalkyl and a group represented by one of the following structural formulas:

46

50. The method of claim 48 wherein:

R3 is 3-isoquinolinyl or 2-benzofuranyl; and

R4 and R5, taken together, form a heterocyclic ring selected from the group consisting of pyrollidine and substituted pyrrolidinyl, indoline, isomers of indoline, substituted indoline, substituted isomers of indoline, tetrahydroisoquinoline, substituted tetrahydroisoquincline, tetrahydroquinoline, substituted tetrahydroquinoline, piperidone, substituted piperidone, piperidine, substituted piperidines, tetrahydro-oxazines and substituted tetrahydro-oxazines.

51. The method of claim 47 wherein R1 is represented by the following structural formula:

47

wherein:

A is selected from the group consisting of an aryl group, a substituted aryl grrouo, a heteroaryl group and a substituted heteroarylgroup; and

n and m are each zero or one.

52. A method of treating an individual suffering from a disease associated with leukocyte infiltration of tissues expressing the molecule MAdCAM-1, comprising administering a therapeutically effective amount of an inhibitor represented by the following structural formula:

R1—X—Y′—Z—R2

wherein:

Y′ is a dipeptide [AA]1-[AA]2 having the sequence Asp-Thr;

X and Z are independently chosen from the group consisting of a covalent bond, an amino acid or a peptide, wherein each amino acid in X and Z is independently selected from the group of naturally occurring amino acids;

R1 is R3—CO—;

R2 is —NR4R5;

R3 is selected from the group consisting of a lower alkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and

R4 and R5 are each independently selected from the group consisting of hydrogen, a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, wherein:

1) R4 and R5 are not both —H; and

2) taken together, R4 and R5 can form a heterocyclic ring; and

taken together, X, Y′ and Z form a peptide containing no more than about fifteen amino acids; and

wherein the peptide formed from X, Y′ and Z is optionally cyclized with the proviso that, if the peptide formed from X, Y′ and Z is cyclized, the nitrogen at the N-terminus of Y′ is not bonded to a glycine or a sarcosine.

53. The method of claim 52 wherein R3 is selected from the group consisting of triphenylmethyl, diphenylmethyl, 3,5-diphenylphenyl, 2-furanyl, 3-furanyl, 9-xanthenemethyl, 2,2,2-triphenylethyl, 2-anthracene, methyl, cyclopentyl, 2-indolyl, 2-indanyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, cyclohexyl, 5-phenylpentyl, 4-isobutyl-&agr;-methylphenylmethyl, 4-biphenylmethyl, &agr;-naphthylmethyl, 4-heptylphenyl, phenylmethyl, trans 2-phenylethenyl and 2,2,3,3-tetramethylcyclopropyl.

54. The method of claim 53 wherein R4 and R5 are each independently selected from the group consisting of —H, 2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, —CH2-2-thienyl, —CH2-3-thienyl, —CH2-2-furanyl, —CH2-3-furanyl, 3,4-dimethoxybenzyl, and isopentyl.

55. The method of claim 52 wherein:

R3 is selected from the group consisting of diphenylmethyl, triphenylmethyl, trans 2-phenyl-ethylenyl, 2-phenyl-ethynyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl and 3-benzothienyl;

R4 is selected from the group consisting of 2-hydroxyethyl, benzyl, 2-benzofuranyl, 3-benzofuranyl, 2-benzothienyl, 3-benzothienyl, —CH2-2-thienyl, —CH2-3-thienyl, —CH2-2-furanyl, —CH2-3-furanyl;

and R5 is —H.

56. The method of claim 55 wherein X and Z are each a covalent bond.

57. The method of claim 52 wherein the peptide formed from X, Y and Z is cyclized.

58. The method of claim 52 wherein the disease is selected from the group consisting of inflammatory bowel disease and insulin-dependent diabetes mellitus.

59. A method of inhibiting the binding of a cell expressing a ligand for MAdCAM-1 on the cell surface to MAdCAM-1 or a portion thereof, comprising contacting the cell with an effective amount of an inhibitor represented by the following structural formula:

R1—X—Y—Z—R2

wherein:

Y is a pentapeptide [AA]1-[AA]2-[AA]3-[AA]4-[AA]5 wherein:

[AA]1 is selected from the group consisting of leucine, cysteine, aspartic acid, glutamic acid, isoleucine, alanine, valine, glycine, N-methylleucine, serine, threonine, ornithine and lysine;

[AA]2 is selected from the group consisting of aspartic acid, glutamic acid, phenylalanine and tyrosine;

[AA]3 is selected from the group consisting of threonine, serine, valine, proline and 4-hydroxyproline;

[AA]4 is selected from the group consisting of serine, cysteine, aspartic acid, glutamic acid, proline, 4-hydroxyproline, threonine, valine, isoleucine, alanine, glycine, ornithine and lysine; and

[AA]5 is selected from the group consisting of leucine, isoleucine, N-methylleucine, threonine, ornithine, serine, valine, alanine, glycine, phenylalanine, cysteine, aspartic acid, glutamic acid and lysine;

X and Z are independently chosen from the group consisting of a covalent bond, an amino acid or a peptide, wherein each amino acid in X and Z is independently selected from the group of naturally occurring amino acids;

R1 is R3—CO—;

R2 is —NR4R5;

R3 is selected from the group consisting of a lower alkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and

R4 and R5 are each independently selected from the group consisting of hydrogen, a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, wherein:

1) R4 and R5 are not both —H; and

2) taken together, R4 and R5 can form a heterocyclic ring; and

taken together, X, Y and Z form a peptide containing no more than about fifteen amino acids; and

wherein optionally the peptide formed from X, Y and Z is cyclized.

60. The method of claim 59 wherein Y is a pentapeptide having the sequence Leu-Asp-Thr-Ser-Leu (SEQ ID NO: 1), Xaa-Asp-Thr-Ser-Leu (SEQ ID NO: 85), Leu-Xaa-Thr-Ser-Leu (SEQ ID NO: 86), Leu-Asp-Xaa-Ser-Leu (SEQ ID NO: 87), Leu-Asp-Thr-Xaa-Leu (SEQ ID NO: 88), or Leu-Asp-Thr-Ser-Xaa (SEQ ID NO: 89), wherein Xaa is a naturally-occurring amino acid.

61. The method of claim 60 wherein the ligand is human &agr;4&bgr;7 integrin.

62. The method of claim 61 wherein the cell is a leukocyte.

63. The method of claim 62 wherein MAdCAM-1 is expressed on the surface of an endothelial cell.

64. The method of claim 59 wherein the peptide formed from X, Y and Z is cyclized.

65. A method of inhibiting the binding of a cell expressing a ligand of MAdCAM-1 to MAdCAM-1 or a portion thereof, comprising contacting the cells with an inhibitory amount of a compound represented by the following structural formula:

R1—X—Y′—Z—R2

wherein:

Y is a dipeptide [AA]1-[AA]2 having the sequence Asp-Thr or a tripeptide [AA]1-[AA]2- [AA]3 having the sequence Leu-Asp-Thr;

X and Z are independently chosen from the group consisting of a covalent bond, an amino acid or a peptide, wherein each amino acid in X and Z is independently selected from the group of naturally occurring amino acids;

R1 is R3—CO—;

R2 is —NR4R5;

R3 is selected from the group consisting of a lower alkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and

R4 and R5 are each independently selected from the group consisting of hydrogen, a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, wherein:

1) R4 and R5 are not both —H; and

2) taken together, R4 and R5 can form a heterocyclic ring; and

taken together, X, Y′ and Z form a peptide containing no more than about fifteen amino acids; and

wherein optionally the peptide formed from X, Y′ and Z is cyclized with the proviso that, if the peptide formed from X, Y′ and Z is cyclized and if Y′ is Asp-Thr, the nitrogen at the N-terminus of Y′ is not bonded to a glycine or a sarcosine.

66. The method of claim 65 wherein the ligand is human &agr;4&bgr;7 integrin.

67. The method of claim 66 wherein the cell is a leukocyte.

68. The method of claim 67 wherein MAdCAM-1 is expressed on the surface of an endothelial cell.

69. The method of claim 65 wherein the peptide formed from X, Y′ and Z is cyclized.

70. A compound represented by the following structural formula:

R1—Y′—R2

wherein:

Y′ is a dipeptide [AA]1-[AA]2 having the sequence Asp-Thr;

R1 is R3—CO—;

R2 is —NR4R5;

R3 is selected from the group consisting of a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group; and

R4 and R5 are each independently selected from the group consisting of hydrogen, a lower alkyl group, a substituted lower alkyl group, an aryl group, a substituted aryl group, a heteroaryl group and a substituted heteroaryl group, wherein:

1) R4 and R5 are not both —H; and

2) taken together, R4 and R5 can feorm a heterocyclic ring.