Combination therapy with cc chemokine receptor 9 (CCR9) inhibitors and Anti-α4β7 integrin blocking antibodies

Abstract

To provide compositions, methods and kits for treating inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis in mammals in need of treatment.SOLUTION: Disclosed is a method comprising conducting a combination therapy with a therapeutically effective amount of chemokine receptor 9 (CCR9)-inhibiting compound and a therapeutically effective amount of an anti-α4β7 integrin antibody such as vedolizumab. The disclosure also provides a kit comprising a chemokine receptor 9 (CCR9)-inhibiting compound and an anti-α4β7 integrin antibody.SELECTED DRAWING: Figure 4C

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 060,454, filed October 6, 2014, the disclosure of which is incorporated by reference in its entirety for all purposes. [Background technology]

[0002] Inflammatory bowel disease (IBD) is a group of chronic inflammatory disorders that affect part or all of the gastrointestinal (GI) tract, including the mouth, esophagus, stomach, small intestine, large intestine (colon), rectum, and anus. IBD includes Crohn's disease (CD), ulcerative colitis (UC), and indeterminate colitis. CD and UC can be distinguished by clinical, endoscopic, and pathological features.

[0003] CD is a chronic inflammatory disease that may involve any part of the gastrointestinal tract. Characteristic symptoms of the disease include severe abdominal pain, frequent diarrhea, rectal bleeding, urgency to defecate, and swelling in the right lower quadrant of the abdomen.

[0004] UC is a chronic, intermittently remitting, inflammatory disease of the large intestine. The disease is characterized by recurrent episodes of inflammation involving predominantly mucosal surface involvement in the rectum and upward into the colon. Acute episodes are characterized by chronic diarrhea or constipation, rectal bleeding, cramps, and abdominal pain.

[0005] IBD is characterized by inflammation and infiltration of blood leukocytes (e.g., lymphocytes, granulocytes, monocytes, and macrophages) into the mucosal or epithelial lining of the intestine. Multiple inflammatory cell types, including lymphocytes, neutrophils, macrophages, and dendritic cells, are involved in IBD. For example, T lymphocytes infiltrate the gastrointestinal mucosa through coordinated interactions between adhesion molecules on their surface and their cognate ligands on the endothelium. For example, the α4β7 integrin expressed on the surface of some T and B lymphocytes induces the migration of these cells by binding to one of its ligands, mucosal addressin cell adhesion molecule 1 (MAdCAM-1), present on gastrointestinal endothelial cells. In IBD, chemokine receptors and ligands (e.g., the receptor CCR9 and its ligand CCL25) also play a role in the migration of inflammatory cells (e.g., effector memory helper T cells) into the intestinal epithelium.

[0006] Current therapies for treating IBD include surgery or the use of anti-tumor necrosis factor (anti-TNFα) antibodies (e.g., infliximab and adalimumab), aminosalicylates, systemic corticosteroids, immunosuppressants (e.g., thiopurines and methotrexate), and combinations thereof. However, some IBD patients do not respond to or cannot tolerate such drug treatments.

[0007] In view of the above, it is apparent that an effective IBD therapeutic regimen capable of blocking multiple pathways and / or multiple cell types involved in lymphocyte infiltration may be useful in the treatment of this disease. The present invention provides treatment using such pharmaceutical compositions and related therapeutic methods. Summary of the Invention [Means for solving the problem]

[0008] In one aspect, the disclosure provides a method of treating or reducing the incidence of inflammatory bowel disease in a mammal, the method comprising administering an appropriate amount of a chemokine receptor CCR9 inhibitor and an anti-α4β7 integrin blocking antibody, in some embodiments, the inflammatory bowel disease is Crohn's disease (CD) or ulcerative colitis (UC).

[0009] In some variations, the chemokine receptor CCR9 inhibitor is a small molecule receptor inhibitor having a molecular weight of less than 1500. The molecular weight of the small molecule receptor inhibitor for CCR9 can be about 1495, 1450, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, or less. In other embodiments, the chemokine receptor CCR9 inhibitor is a small molecule receptor inhibitor having a molecular weight of less than 750. The molecular weight of the small molecule receptor inhibitor for CCR9 may be about 745, 700, 650, 600, 550, 500, 450, 400, 350, 300, or less.

[0010] In some embodiments, the small molecule CCR9 inhibitor described herein may be a compound represented by formula (I) or a salt thereof: [ka] (In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , L., A. 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , and A 8 is defined below).

[0011] In some embodiments, the anti-α4β7 integrin blocking antibody is vedolizumab (ENYVIO®), or a biosimilar, biobetter, or bioequivalent thereof. In other embodiments, the anti-α4β7 integrin blocking antibody is vedolizumab (ENYVIO®).

[0012] In some embodiments, the chemokine receptor CCR9 inhibitor and the anti-α4β7 integrin blocking antibody are administered in a combined formulation. In other embodiments, the chemokine receptor CCR9 inhibitor and the anti-α4β7 integrin blocking antibody are administered sequentially. In yet other embodiments, the chemokine receptor CCR9 inhibitor is administered prior to the anti-α4β7 integrin blocking antibody. In another embodiment, the chemokine receptor CCR9 inhibitor is administered after the anti-α4β7 integrin blocking antibody.

[0013] In some embodiments, the chemokine receptor CCR9 inhibitor is a compound having the formula: [ka]

[0014] In another aspect, the disclosure provides a composition for treating or reducing the incidence of inflammatory bowel disease in a mammal, comprising a therapeutically effective amount of a chemokine receptor CCR9 inhibitor, a therapeutically effective amount of an anti-α4β7 integrin blocking antibody, and a pharma- ceutically acceptable carrier or excipient.

[0015] In some variations, the inflammatory bowel disease is Crohn's disease (CD) or ulcerative colitis (UC).

[0016] In some embodiments, the chemokine receptor CCR9 inhibitor is a small molecule receptor inhibitor having a molecular weight of less than 1500. In other embodiments, the chemokine receptor CCR9 inhibitor is a small molecule receptor inhibitor having a molecular weight of less than 750. The chemokine receptor CCR9 inhibitor compound can be a compound represented by Formula (I) or a salt thereof as described herein.

[0017] In some embodiments, the anti-α4β7 integrin blocking antibody is vedolizumab (ENYVIO®), or a biosimilar, biobetter, or bioequivalent thereof. In other embodiments, the anti-α4β7 integrin blocking antibody is vedolizumab (ENYVIO®).

[0018] In yet another aspect, the present disclosure provides a kit for treating or reducing the incidence of inflammatory bowel disease in a mammal, comprising a therapeutically effective amount of a chemokine receptor CCR9 inhibitor and a therapeutically effective amount of an anti-α4β7 integrin blocking antibody, and instructions for effective administration.

[0019] In some embodiments, the chemokine receptor CCR9 inhibitor and the anti-α4β7 integrin blocking antibody are formulated for sequential administration, while in other embodiments, the chemokine receptor CCR9 inhibitor and the anti-α4β7 integrin blocking antibody are formulated for simultaneous administration.

[0020] In some embodiments, the chemokine receptor CCR9 inhibitor is a small molecule receptor inhibitor having a molecular weight of less than 1500. In other embodiments, the chemokine receptor CCR9 inhibitor is a small molecule receptor inhibitor having a molecular weight of less than 750. The chemokine receptor CCR9 inhibitor compound can be a compound of Formula (I) or a salt thereof as described herein. Optionally, the small molecule receptor inhibitor is versanone (Traficet-EN™) or CCX507.

[0021] In some embodiments, the anti-α4β7 integrin blocking antibody is vedolizumab (ENYVIO®), or a biosimilar, biobetter, or bioequivalent thereof. In other embodiments, the anti-α4β7 integrin blocking antibody is vedolizumab (ENYVIO®).

[0022] Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description and drawings. [Brief description of the drawings]

[0023] [Figure 1A] Figure 1A and Figure 1B show that a small molecule inhibitor of CCR9 (CCX507) restricts human T cells differentiated with retinoic acid from binding to MAdCAM-1. Figure 1A shows that human peripheral blood mononuclear cells (PBMCs) were activated by CD3ε and CD28 in the presence of retinoic acid and IL-12. Expression of gut-acting factors CCR9 and α4β7 are shown as two-parameter dot plots. The percentage of CCR9-positive α4β7-positive cells is shown. [Figure 1B] Figure 1A and Figure 1B show that a small molecule inhibitor of CCR9 (CCX507) limits MAdCAM-1 binding in human T cells differentiated with retinoic acid. Figure 1B shows data from a static binding assay of ex vivo differentiated human CCR9+·α4β7+ T cells. Briefly, CCR9+·α4β7+ cells were mixed with hCCL25 (500 nM) in the presence or absence of CCX507 (1 μM) and added to MAdCAM-1-Fc-coated plates. The number of cells adhering to MAdCAM-1 in each condition was quantified using CyQUANT® and expressed as relative fluorescence units (RFU). [Figure 2A] Figures 2A, 2B, and 2C show the pharmacodynamic efficacy of CCX507 in vivo in a mouse model of adoptive T cell transfer. Figure 2A shows the experimental design used to confirm the pharmacodynamic range of CCX507 in vivo in a short-term T cell delivery model. [Figure 2B] Figure 2A, Figure 2B, and Figure 2C show the pharmacodynamic efficacy of CCX507 in vivo in a mouse model of adoptive T cell transfer. Figure 2B shows bar graphs of the number of OT-I-derived CD8 positive intraepithelial lymphocytes (IELs) in the presence of CCX507 (5-30 mg / kg) and Figure 2C shows bar graphs of the number of OT-I-derived CD8 positive intraepithelial lymphocytes (IELs) in the presence of an anti-α4β7 integrin blocking antibody. [Figure 2C]Figure 2A, Figure 2B, and Figure 2C show the pharmacodynamic efficacy of CCX507 in vivo in a mouse model of adoptive T cell transfer. Figure 2B shows bar graphs of the number of OT-I-derived CD8 positive intraepithelial lymphocytes (IELs) in the presence of CCX507 (5-30 mg / kg) and Figure 2C shows bar graphs of the number of OT-I-derived CD8 positive intraepithelial lymphocytes (IELs) in the presence of an anti-α4β7 integrin blocking antibody. [Diagram 3] Figure 3 shows the relationship between human CCR9 gene expression in the ileum and colon. Biopsy samples were obtained from patients with Crohn's disease. Normalized gene expression was derived from samples from the terminal ileum and colon. Figure 3 shows a color-coded diagram representing the expression levels of selected genes relative to the expression levels of CCR9. [Figure 4A] Figures 4A, 4B, 4C, and 4D show that the combination of CCX507 with an anti-α4β7 integrin blocking antibody provided superior protection from colitis compared to monotherapy treatment regimens. Figure 4A shows the dosing regimen of an anti-α4β7 integrin blocking antibody and an anti-TNFα antibody. [Figure 4B] Figures 4A, 4B, 4C, and 4D show that the combination of CCX507 and anti-α4β7 integrin blocking antibody provided superior protection from colitis compared to monotherapy treatment regimens. Figure 4B shows representative photographs of the colons of wild-type mice (FVB) and mice given vehicle control (1% HPMC), rat IgG2a isotype control, CCX507, anti-α4β7 integrin blocking antibody, and the combination of CCX507 and anti-α4β7 integrin blocking antibody. [Figure 4C] Figures 4A, 4B, 4C, and 4D show that the combination of CCX507 and an anti-α4β7 integrin blocking antibody provided superior protection from colitis compared to monotherapy treatment regimens. Scatter plots of quantified colon-to-body weight ratios are shown for CCX507 and an anti-α4β7 integrin blocking antibody (Figure 4C) and CCX507 and an anti-TNFα blocking antibody (Figure 4D). [Figure 4D]Figures 4A, 4B, 4C, and 4D show that the combination of CCX507 and an anti-α4β7 integrin blocking antibody provided superior protection from colitis compared to monotherapy treatment regimens. Scatter plots of quantified colon-to-body weight ratios are shown for CCX507 and an anti-α4β7 integrin blocking antibody (Figure 4C) and CCX507 and an anti-TNFα blocking antibody (Figure 4D). [Figure 5A] Figures 5A and 5B show the results of histological analysis of mouse colon tissue. Figure 5A shows representative images of both the proximal and distal colons of individual mice, representing the average histopathological score for each group. The right panel shows the combination of CCX507 and anti-α4β7 antibody. [Figure 5B] Figures 5A and 5B show the results of histological analysis of mouse colon tissue, with Figure 5B showing the combined histological scores of all mice tested. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] (I. Introduction) The present disclosure is based in part on the unexpected discovery that combination therapy with a CCR9 inhibitor (e.g., a small molecule inhibitor of CCR9) and an antibody against α4β7 integrin may act synergistically in the treatment of inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, indeterminate colitis, etc.). Described herein are methods, compositions, and kits for treating IBD in a subject (e.g., a human or animal subject) in need of such treatment. In some embodiments, the methods include administering to an IBD patient therapeutically effective amounts of CCX507 and vedolizumab to induce a clinical response or maintain clinical remission in the patient.

[0025] (II. Definition) When describing the compounds, compositions, methods and processes of this invention, the following terms have the following meanings, unless otherwise indicated.

[0026] As used herein, "a," "an," or "the" includes aspects having one or more of a given component. For example, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, reference to "the agent" includes reference to one or more agents known to those of skill in the art, and so forth.

[0027] The term "about" is generally intended to mean an acceptable degree of error for a measured quantity given the nature or precision of the measurement. An example of a typical degree of error is within 20 percent (%) (within 10% being preferred, and within 5% being more preferred) of a given value or range of values. Alternatively, particularly in biological systems, the term "about" may mean a value within 10-fold (within 5-fold being preferred, and within 2-fold being more preferred) of a given value. Numerical quantities provided herein are approximate quantities unless otherwise specified, i.e., the term "about" can be inferred when not specified.

[0028] The term "inflammatory bowel disease" or "IBD" includes gastrointestinal disorders such as, for example, Crohn's disease (CD), ulcerative colitis (UC), indeterminate colitis (IC), and IBD where either CD or UC is undetermined ("indeterminate"). Inflammatory bowel disease (e.g., CD, UC, IC, indeterminate) is distinct from all other gastrointestinal disorders, syndromes, or conditions, such as irritable bowel syndrome (IBS). Examples of IBD-related disorders include collagenous colitis and lymphocytic colitis.

[0029] The term "ulcerative colitis" or "UC" refers to a chronic, intermittent relapsing inflammatory bowel disease (IBD) of the colon or large intestine, characterized by lesions of the mucosal surface that extend to the rectum and progress upward. Different types of ulcerative colitis are classified according to the location and extent of inflammation. Examples of UC include, but are not limited to, ulcerative proctitis, proctosigmoiditis, left-sided colitis, and ulcerative pancolitis.

[0030] The term "Crohn's disease" or "CD" refers to a chronic inflammatory disorder that may involve any part of the gastrointestinal tract. The distal small intestine (i.e., ileum) and cecum are commonly affected. In other cases, the disease may be limited to the small intestine, colon, or anorectal region. CD may also involve the duodenum and stomach, and more rarely, the esophagus and mouth. Examples of UC include, but are not limited to, ileitis, gastroduodenal Crohn's disease, jejunoileitis, and colonic Crohn's disease (granulomatous).

[0031] The terms "subject," "individual," or "patient" refer to animals such as mammals, including but not limited to primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc.

[0032] The term "CC chemokine receptor 9," "CCR9," or "chemokine receptor CCR9" refers to the receptor for the chemokine CCL25 (also known as TECK, SCYA25). The polypeptide sequence of human CCR9 is set forth, for example, in GenBank Accession Nos. NP_001243298, NP_006632, NP_112477, and XP_011531614. The mRNA (coding) sequence of human CCR9 is set forth, for example, in GenBank Accession Nos. NM_001256369, NM_006641, NM_031200, and XM_011533312.

[0033] The terms "CC chemokine receptor 9 inhibitor", "CCR9 inhibitor" or "chemokine receptor CCR9 inhibitor" refer to inhibitors or antagonists of the CCR9 receptor polypeptide, variants thereof, or fragments thereof.

[0034] The term "small molecule inhibitor" refers to a small molecule or low molecular weight organic compound that inactivates, inhibits, or antagonizes a target molecule, biomolecule, protein, or other biological entity.

[0035] The term "α4β7 integrin" refers to a heterodimeric integrin molecule having two different chains, such as an α4 chain and a β7 chain. The polypeptide sequence of the human integrin α4 chain is described, for example, in GenBank Accession No. NP_000876. The mRNA (coding) sequence of the human integrin α4 chain is described, for example, in GenBank Accession No. NM_000885. The polypeptide sequence of the human integrin β7 chain is described, for example, in GenBank Accession Nos. NP_000880, XP_005268908, and XP_005268909. The mRNA (coding) sequence of the human integrin β7 chain is described, for example, in GenBank Accession Nos. NM_000889, XM_05268851, and XM_05268852. The α4β7 integrin is also referred to as LPAM.

[0036] The term "anti-α4β7 integrin blocking antibody" or "anti-α4β7 integrin neutralizing antibody" refers to an antibody or fragment thereof that specifically binds to an α4β7 integrin (α4β7 integrin heterodimer) polypeptide or a fragment thereof. In some cases, the anti-α4β7 integrin blocking antibody blocks the interaction of the α4β7 integrin with any one of its ligands.

[0037] The term "biosimilar" refers to a biological product that is highly similar to an FDA-approved biological product (reference product) and that has no clinically meaningful differences from the reference product in terms of pharmacokinetics, safety, or efficacy.

[0038] The term "bioequivalent" refers to a biological product that is pharmacologic equivalent to and has similar bioavailability to an FDA-approved biological product (reference product). For example, according to the FDA, the term "bioequivalent" is defined as "the absence of significant differences in the proportion or extent to which the active ingredient or active moiety of a pharmaceutical equivalent or pharmaceutical substitute is available at the site of drug action when administered at the same molar dose under similar conditions in appropriately designed studies" (United States Food and Drug Administration, "Guidance for Industry: Bioavailability and Bioequivalence Studies for Orally Administered Drug Products - General Considerations," 2003, Center for Drug Evaluation and Research).

[0039] The term "biobetter" refers to a biological product that is in the same category as an FDA-approved biological product (the reference product) but is not identical to it, and that has improved safety, efficacy, stability, etc. compared to the reference product.

[0040] The term "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to ameliorate a condition or symptom of interest. For example, for a given parameter, a therapeutically effective amount may exhibit an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy may also be expressed as "-fold" more or less effective. For example, a therapeutically effective amount may be at least 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effective than a control.

[0041] The term "administering" and its derivatives refer to methods that can be used to deliver an agent or composition to the site of desired biological action. Such methods include, but are not limited to, parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intranasal, intravitreal, intravenous, local injection, etc.), transmucosal injection, oral administration, administration of a suppository, and topical administration. Those skilled in the art will be aware of additional methods of administering a therapeutically effective amount of a compound of the present invention to prevent or reduce one or more symptoms associated with a disease.

[0042] The term "treating" refers to the treatment of a disease or medical condition (such as inflammation) in a patient, such as a mammal (especially a human or animal), including ameliorating the patient's disease or medical condition (i.e., eliminating or reversing the disease or medical condition); inhibiting the patient's disease or medical condition (i.e., slowing or arresting the onset of the disease or medical condition); or alleviating the symptoms of the patient's disease or medical condition. The term encompasses prophylactic treatment of disease to prevent or reduce the risk of acquiring or developing a particular disease, or to prevent or reduce the risk of recurrence of the disease.

[0043] "Alkyl," by itself or as part of another substituent, has a specified number of carbon atoms (i.e., C 1~8means 1 to 8 carbon atoms), linear, cyclic, branched, or a combination thereof. The term "cycloalkyl", by itself or as part of another substituent, refers to a cyclic alkyl group having a specified number of carbon atoms and is a subset of the term "alkyl". Another subset of the term "alkyl" includes "straight-chain" and "branched-chain" alkyl groups, which refer to two different types of non-cyclic alkyl groups. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, cyclopentyl, (cyclohexyl)methyl, cyclopropylmethyl, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and the like. In the examples listed herein, the examples of alkyl, methyl, ethyl, n-propyl, and n-butyl, are also examples of "straight-chain" alkyl groups. Similarly, isopropyl and t-butyl are also examples of "branched-chain" alkyl groups. Cyclopentyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, bicyclo[2.2.1]heptane, and bicyclo[2.2.2]octane are examples of "cycloalkyl" groups. In some embodiments, cyclopropyl is used as a bridging group between two other moieties and may be represented as -CH(CH2)CH-. An "alkyl group" may be substituted or unsubstituted unless otherwise indicated. Examples of substituted alkyls include haloalkyls, thioalkyls, aminoalkyls, and the like. Further examples of suitable substituted alkyls include, but are not limited to, hydroxyisopropyl, -C(CH3)2-OH, aminomethyl, 2-nitroethyl, 4-cyanobutyl, 2,3-dichloropentyl, 3-hydroxy-5-carboxyhexyl, 2-aminoethyl, pentachloroethyl, trifluoromethyl, 2-diethylaminoethyl, 2-dimethylaminopropyl, ethoxycarbonylmethyl, methanylsulfanylmethyl, methoxymethyl, 3-hydroxypentyl, 2-carboxybutyl, 4-chlorobutyl, and pentafluoroethyl.Suitable substituents for substituted alkyl include halogen, -CN, -CO2R', -C(O)R', -C(O)NR'R'', oxo (=O or -O. - ), -OR', -OC(O)R', -OC(O)NR'R''-NO2, -NR'C(O)R'', -NR'''C(O)NR'R'', -NR'R'', -NR'CO2R'', -NR'S(O)R'', -NR'S(O)2R''', -NR'''S(O)NR'R'', -NR'''S(O)2NR'R'', -SR', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NR'-C(NHR'')=NR''', -SiR'R''R'''', -OSiR'R''R'''', -N3, substituted or unsubstituted C 6~10 Examples of substituted alkyl include aryl, substituted or unsubstituted 5- to 10-membered heteroaryl, and substituted or unsubstituted 3- to 10-membered heterocyclyl. The number of possible substituents ranges from 0 to (2m'+1), where m' is the total number of carbon atoms in the radical. For substituted alkyl, R', R'', and R''' are each independently hydrogen, substituted or unsubstituted C 1~8 Alkyl, substituted or unsubstituted C 2~8 Alkenyl, substituted or unsubstituted C 2~8 It refers to various groups such as alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryloxyalkyl, etc. When R' and R'' are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring (e.g., -NR'R'' includes 1-pyrrolidinyl and 4-morpholinyl). R' and R'', R'' and R''', or R' and R''', together with the atom to which they are attached, can also form a substituted or unsubstituted 5-, 6-, or 7-membered ring.

[0044] "Alkoxy" refers to -O-alkyl. Examples of alkoxy groups include methoxy, ethoxy, n-propoxy, and the like.

[0045] "Alkenyl" refers to an unsaturated hydrocarbon group that may be linear, cyclic, branched, or a combination thereof. Alkenyl groups having 2 to 8 carbon atoms are preferred. Alkenyl groups may contain 1, 2, or 3 carbon-carbon double bonds. Examples of alkenyl groups include ethenyl, n-propenyl, isopropenyl, n-but-2-enyl, n-hex-3-enyl, cyclohexenyl, cyclopentenyl, and the like. Alkenyl groups may be substituted or unsubstituted unless otherwise indicated.

[0046] "Alkynyl" refers to an unsaturated hydrocarbon group that may be linear, cyclic, branched, or a combination thereof. Alkynyl groups having 2 to 8 carbon atoms are preferred. Alkynyl groups may contain 1, 2, or 3 carbon-carbon triple bonds. Examples of alkynyl groups include ethynyl, n-propynyl, n-but-2-ynyl, n-hex-3-ynyl, and the like. Alkynyl groups may be substituted or unsubstituted unless otherwise indicated.

[0047] "Alkylamino" refers to -N(alkyl)2 or -NH(alkyl). When an alkylamino group contains two alkyl groups, the alkyl groups may together form a carbocyclic or heterocyclic ring. It is noted that the alkyl groups of an alkylamino group may be substituted or unsubstituted. Examples of alkylamino groups include methylamino, tert-butylamino, dimethylamino, diisopropylamino, morpholino, and the like.

[0048] "Aminoalkyl" refers to a mono- or polyaminoalkyl group (most commonly substituted with one or two amino groups) as a substituted alkyl group. Examples include aminomethyl, 2-aminoethyl, 2-diethylaminoethyl, and the like.

[0049] "Aryl" refers to a polyunsaturated aromatic hydrocarbon group having one ring (bicyclic) or multiple rings (preferably bicyclic) which may be fused or covalently linked. Aryl groups having 6 to 10 carbon atoms are preferred, e.g., C 6~10 Examples of aryl groups include phenyl, naphthalene-1-yl, naphthalene-2-yl, biphenyl, and the like. An aryl group may be substituted or unsubstituted unless otherwise indicated. A substituted aryl may be substituted with one or more substituents. Suitable substituents for an aryl include substituted or unsubstituted C 1~8 Included are the substituents set forth above for alkyl and substituted alkyl.

[0050] "Halo" or "halogen," by itself or as part of a substituent, refers to a chlorine, bromine, iodine, or fluorine atom.

[0051] "Haloalkyl" refers to a substituted alkyl group, a monohaloalkyl group or a polyhaloalkyl group, most commonly substituted with 1 to 3 halogen atoms. Examples include 1-chloroethyl, 3-bromopropyl, trifluoromethyl, and the like.

[0052] "Heterocyclyl" refers to a saturated or unsaturated non-aromatic group having at least one (typically 1-5) heteroatom selected from nitrogen, oxygen, and sulfur. These groups preferably contain 0-5 nitrogen atoms, 0-2 sulfur atoms, and 0-2 oxygen atoms, and more preferably 0-3 nitrogen atoms, 0-1 sulfur atoms, and 0-1 oxygen atoms. Examples of heterocyclic groups include pyrrolidine, piperidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-dioxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, etc. Preferred heterocyclic groups are monocyclic, but may be fused or covalently linked to an aryl or heteroaryl ring system.

[0053] An exemplary heterocyclic group may be a group represented by formula (AA): [ka]

[0054] Here, formula (AA) is M 1 or M 2 The bond is made via the free valence of M. 1 , O, NR e , or S(O) l represents M 2 is CR f R g , O, S(O) l , or N.R. e where R f , R g , or R e Remove one and get M 1 or M 2 (For example, CR f , C.R. g, or N), l is 0, 1, or 2, j is 1, 2, or 3, and k is 1, 2, or 3, where j+k is 3, 4, or 5. a , R b , R c , R d , R e , R f , and R g is hydrogen, halogen, substituted or unsubstituted C 1~8 Alkyl, substituted or unsubstituted C 2~8 Alkenyl, substituted or unsubstituted C 2~8 Alkynyl, -COR h , -CO2R h , -CONR h R i , -NR h COR i , -SO2R h -SO2NR h R i , -NR h SO2R i -NR h R i , -OR h , -SiR h R i R j , -OSiR h R i R j , -Q 1 COR h , -Q 1 CO2R h , -Q 1 CONR h R i , -Q 1 NR h COR i , -Q 1 SO2R h , -Q 1 SO2NR h R i , -Q 1 NR h SO2R i , -Q 1 NR h R i , -Q 1 OR h (Here, Q1 is C 1~4 Alkylene, C 2~4 Alkenylene, and C 2~4 alkynylene, R h , R i , and R j is hydrogen and C 1~8 alkyl), where the substituent R a , R b , R c , R d , R e , R f , R g , R h , R i and each aliphatic portion of R is halogen, -OH, -OR n , -OC(O)NHR n , -OC(O)NR n R o , -SH, -SR n , -S(O)R n , -S(O)2R n -S(O)2NHR n , -S(O)NR n R o , -NHS(O)2R n , -NR n S(O)2R o , -C(O)NH2, -C(O)NHR n , -C(O)NR n R o , -C(O)R n , -NHC(O)R o , -NR n C(O)R o , -NHC(O)NH2, -NR n C(O)NH2, -NR n C(O)NHR o , -NHC(O)NHR n , -NR n C(O)NR o R p , -NHC(O)NR n R o , -CO2H, -CO2R n , -NHCO2R n , -NRn CO2R o , -CN, -NO2, -NH2, -NHR n , -NR n R o , -NR n S(O)NH2, and -NR n S(O)2NHR o (where R n , R o , and R p are independently unsubstituted C 1~8 alkyl). a , R b , R c , R d , R e , R f , and R g Any two of may together form a bridged or spiro ring system.

[0055] R a +R b +R c +R d Preferably, the number of groups other than hydrogen in R is 0, 1, or 2. More preferably, R a , R b , R c , R d , R e , R f , and R g is hydrogen, halogen, substituted or unsubstituted C 1~8 Alkyl, -C(O)R h , -CO2R h , -C(O)NR h R h , -NR h COR i , -SO2R h , -SO2NR h R i , -NSO2R h R i , -NR h R i , and -OR h where R h and R i is hydrogen and unsubstituted C1~8 alkyl; a , R b , R c , R d , R e , R f , and R g Each aliphatic portion of n , -OC(O)NHR n , -OC(O)NR n R o , -SH, -SR n , -S(O)R o , -SO2R n , -SO2NH2, -SO2NHR n , -SO2NR n OR o , -NHSO2R n , -NR n SO2R o , -C(O)NH2, -C(O)NHR n , -C(O)NR n R o , -C(O)R n , -NHC(O)R n , -NR n C(O)R o , -NHC(O)NH2, -NR n C(O)NH2, -NR n C(O)NHR o , -NHC(O)NHR n , -NR n C(O)NR n R o , -NHC(O)NR n R o , -CO2H, -CO2R n , -NHCO2R n , -NR n CO2R o , -CN, -NO2, -NH2, -NHR n , -NR n R o , -NR n S(O)NH2, and -NR n S(O)2NHR o (where R n , R o , and R pare independently unsubstituted C 1~8 and alkyl.

[0056] R a , R b , R c , R d , R e , R f , and R g are independently hydrogen or C 1~4 More preferably, R is alkyl. a , R b , R c , R d , R e , R f , and R g At least three of them are hydrogen.

[0057] A "heteroaryl" is an aromatic group having at least one heteroatom. Examples include pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzothiazolyl, benzofuranyl, benzothienyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, and thienyl. Preferred heteroaryl groups are those having at least one aryl ring nitrogen atom, such as quinolinyl, quinoxalinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzothiazolyl, indolyl, quinolyl, isoquinolyl, etc. Preferred 6-membered heteroaryl systems include pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, etc. Preferred 5-membered heteroaryl systems include isothiazolyl, pyrazolyl, imidazolyl, thienyl, furyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, thiazolyl, etc.

[0058] Heterocyclyl and heteroaryl can be attached at any available carbon atom or heteroatom in the ring. Each heterocyclyl and heteroaryl may have one or more rings. When multiple rings are present, they may be fused, i.e. covalently linked. Each heterocyclyl and heteroaryl must have at least one (typically 1-5) heteroatom selected from nitrogen, oxygen, and sulfur. These groups preferably have 0-5 nitrogen atoms, 0-2 sulfur atoms, and 0-2 oxygen atoms, and more preferably 0-3 nitrogen atoms, 0-1 sulfur atoms, and 0-1 oxygen atoms. Heterocyclyl and heteroaryl groups may be substituted or unsubstituted unless otherwise indicated. In the case of substituted groups, the substitution may be at a carbon atom or a heteroatom. For example, the substitution may be oxo (=O or -O - ), the resulting group is a carbonyl (-C(O)-) or an N-oxide (-N + -O - ) or -S(O)- or -S(O)2-.

[0059] Suitable substituents for substituted alkyl, alkenyl, and alkynyl include halogen, -CN, -COR', -C(O)R', -C(O)NR'R'', oxo (=O or -O - ), -OR', -OC(O)R', -OC(O)NR'R''-NO2, -NR'C(O)R'', -NR'''C(O)NR'R'', -NR'R'', -NR'CO2R'', -NR'S(O)2R''', -SR', -S(O)R', -S(O)2R', -S(O)2NR'R'', -SiR'R''R''', -N3, substituted or unsubstituted C 6~10 Examples of the heteroaryl include aryl, substituted or unsubstituted 5- to 10-membered heteroaryl, and substituted or unsubstituted 3- to 10-membered heterocyclyl, and the number of substituents ranges from 0 to (2m'+1), where m' is the total number of carbon atoms contained in the radical.

[0060] Suitable substituents for substituted aryl, substituted heteroaryl, and substituted heterocyclyl include halogen, -CN, -COR', -C(O)R', -C(O)NR'R'', oxo (=O or -O - ), -OR', -OC(O)R', -OC(O)NR'R''-NO2, -NR'C(O)R'', -NR'C(O)NR''R''', -NR'R'', -NR'CO2R'', -NR'S(O)2R'', -SR', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NR'-C(NHR'')=NR''', -SiR'R''R''', -N3, substituted or unsubstituted C 1~8 Alkyl, substituted or unsubstituted C 2~8 Alkenyl, substituted or unsubstituted C 2~8 Alkynyl, substituted or unsubstituted C 6~10 Examples include aryl, substituted or unsubstituted 5- to 10-membered heteroaryl, and substituted or unsubstituted 3- to 10-membered heterocyclyl. The number of possible substituents ranges from 0 to the total number of free valences on the aromatic ring system.

[0061] As used above, R', R'', and R''' are each independently hydrogen, substituted or unsubstituted C 1~8 Alkyl, substituted or unsubstituted C 2~8 Alkenyl, substituted or unsubstituted C 2~8 It refers to various groups such as alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted aryloxyalkyl, etc. When R' and R'' are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring (e.g., -NR'R'' includes 1-pyrrolidinyl and 4-morpholinyl). R' and R'', R'' and R''', or R' and R''', together with the atom to which they are attached, can also form a substituted or unsubstituted 5-, 6-, or 7-membered ring.

[0062] Two of the substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be of the formula -TC(O)-(CH2) q -U- (wherein T and U are independently -NR''''-, -O-, -CH2- or a single bond, and q is an integer from 0 to 2) or a substituent of the formula -A'-(CH2) r A' and B' are independently -CH2-, -O-, -NR''''-, -S-, -S(O)-, -S(O)2-, -S(O)2NR''''-, or a single bond, and r is an integer from 1 to 3. One of the single bonds in the new ring thus formed may optionally be replaced by a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by a substituent of the formula -(CH2) s -X-(CH2) t - (wherein s and t are independently an integer of 0 to 3, and X is -O-, -NR''''-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-). The substituent R'''' of -NR''''- and -S(O)2NR'''' is hydrogen or unsubstituted C 1~8 It is an alkyl.

[0063] "Heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

[0064] A "pharmaceutical acceptable" carrier, diluent, or excipient is a carrier, diluent, or excipient that is compatible with other ingredients in the formulation and is not deleterious to the patient.

[0065] "Pharmaceutically acceptable salt" refers to a salt that is acceptable for administration to a patient, such as a mammal (e.g., a salt that is acceptable for safety in a mammal for a given dosing regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids, depending on the particular substituents found on the compounds described herein. When the compounds of the present invention have relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of the compound with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Salts derived from pharmaceutically acceptable inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, ferric salts, ferrous salts, lithium salts, magnesium salts, manganous salts, manganous salts, potassium salts, sodium salts, zinc salts, and the like. Salts derived from pharma- ceutically acceptable organic bases include salts of primary, secondary, tertiary, and quaternary amines (such as substituted, cyclic, and naturally occurring amines), such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. When a compound of the present invention has a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of the compound with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.Salts derived from pharma- ceutically acceptable acids include acetate, ascorbate, benzenesulfonate, benzoate, camphorsulfonate, citrate, ethanesulfonate, fumarate, gluconate, glucuronate, glutamate, hippurate, hydrobromide, hydrochloride, isethionate, lactate, lactobionate, maleate, malate, mandelate, methanesulfonate, mucate, naphthalenesulfonate, nicotinate, nitrate, pamoate, pantothenate, phosphate, succinate, sulfate, tartrate, p-toluenesulfonate, and the like.

[0066] Also included are salts of amino acids, such as arginate, and salts of organic acids, such as glucuronic acid or galacturonic acid (see, e.g., Berge, SM, et al, "Pharmaceutical Salts", J. Pharmaceutical Science, 1977, 66:1-19).Certain compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0067] Neutral forms of the compounds can be produced by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent compound forms differ from the various salt forms described above in certain physical properties, such as solubility in polar solvents, but are otherwise equivalent to the parent compound forms for purposes of the present invention.

[0068] "Salt thereof" refers to a compound formed when a hydrogen of an acid is replaced by a cation, such as a metallic cation or an organic cation. Preferably, the salt is a pharma- ceutically acceptable salt, although this is not required for salts of intermediate compounds that are not intended for administration to a patient.

[0069] In addition to salt forms, the present invention provides compounds in prodrug form. Prodrugs are often useful because in some circumstances they may be easier to administer than the parent drug. For example, a prodrug may be bioavailable by oral administration while the parent drug is not. A prodrug may also be more soluble in pharmaceutical compositions than the parent drug. Various types of prodrug derivatives are known in the art, including those that utilize hydrolytic cleavage or oxidative activation of the prodrug. A non-limiting example of a prodrug is a compound of the present invention that is administered as an ester ("prodrug") but is metabolically hydrolyzed to the carboxylic acid, which is the active entity. Further examples include peptidyl derivatives of the compounds of the present invention.

[0070] Prodrugs of the compounds described herein are compounds that undergo easy chemical changes under physiological conditions to become the compounds of the present invention. Also, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an in vitro environment. For example, when a prodrug is placed in a reservoir of a transdermal patch with a suitable enzyme or chemical reagent, it can be slowly converted to the compounds of the present invention.

[0071] Prodrugs can be prepared by modifying functional groups present in a compound in such a way that the modification is cleaved, either routinely or in vivo, to the parent compound. Prodrugs include compounds in which a hydroxyl, amino, sulfhydryl, or carboxyl group is bonded to any group that, upon administration to a mammalian subject, is cleaved to form a free hydroxyl, amino, sulfhydryl, or carboxyl group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol and amine functional groups contained in the compounds of the present invention. The preparation, selection and use of prodrugs are described in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," ACS Symposium Series, Vol. 14; "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985; and Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, the entire contents of each of which are incorporated by reference.

[0072] The compounds of the present invention may exist in the form of their pharma- ceutically acceptable metabolites. The term "metabolite" refers to a pharma-ceutically acceptable metabolic derivative of the compounds of the present invention (or their salts). In some aspects, metabolites may be functional derivatives of compounds that can be readily converted to active compounds in vivo. In other aspects, metabolites may be active compounds.

[0073] The term "acid isostere", unless otherwise specified, refers to a group that has an acidic functionality and can replace a carboxylic acid, with steric and electronic properties that provide a level of activity (or other characteristic of the compound, such as solubility) similar to that of the carboxylic acid. Representative acid isosteres include hydroxamic acids, sulfonic acids, sulfinic acids, sulfonamides, acylsulfonamides, phosphonic acids, phosphinic acids, phosphoric acids, tetrazoles, and oxooxadiazoles.

[0074] Certain compounds of the present invention can exist in unsolvated form or in solvated form, such as hydrated form. In general, both solvated and unsolvated forms are intended to be included within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or non-crystalline forms (i.e., polymorphs). In general, all physical forms are equivalent for the use contemplated by the present invention and are intended to be within the scope of the present invention.

[0075] Certain compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds. The racemates, diastereomers, geometric isomers and individual isomers (e.g., separate enantiomers) are all intended to be encompassed within the scope of the present invention. Compounds of the present invention may further contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds. For example, compounds may be enriched in tritium ( 3 H), Iodine-125( 125 I), or carbon-14 ( 14 Any isotopic variation of the compounds of the present invention, including but not limited to radioisotopes, is intended to be encompassed within the scope of the present invention.

[0076] The compound of the present invention may contain a detectable label. A detectable label is a group that can be detected at low concentrations (usually submicromolar, likely subnanomolar, and in some cases subpicomolar) and can be easily distinguished from other molecules by differences in molecular properties (e.g., molecular weight, mass-to-charge ratio, radioactivity, redox potential, luminescence, fluorescence, electromagnetic properties, binding properties, etc.). Detectable labels can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, magnetic, electromagnetic, optical or chemical means, etc.

[0077] A wide variety of detectable labels are within the scope of the present invention, including hapten labels (e.g., biotin, or labels used in conjunction with a detectable antibody, such as a horseradish peroxidase antibody); mass tag labels (e.g., stable isotope labels); radioisotope labels (e.g., 3 H, 125 I, 35 S, 14 C, or 32, P, and the like; metal chelate labels; luminescent labels (generally with a quantum yield greater than 0.1), such as fluorescent labels (fluorescein, isothiocyanate, Texas Red, rhodamine, green fluorescent protein, and the like), phosphorescent labels, and chemiluminescent labels; electroactive labels and electron transfer labels; labels with enzyme modifiers (such as coenzymes, organometallic catalysts, horseradish peroxidase, alkaline phosphatase, and other labels commonly used in enzyme-linked immunosorbent assays (ELISAs)); photosensitizer labels; magnetic bead labels such as Dynabeads®; colorimetric labels (such as colloidal gold, silver, selenium, or other metals, and metal sol labels (see U.S. Pat. No. 5,120,643, the entire contents of which are incorporated herein by reference for all purposes), or labels with colored glass beads or colored plastic (polystyrene, polypropylene, latex, and the like) beads); and carbon black labels. Patents which teach the use of these detectable labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, 4,366,241, 6,312,914, 5,990,479, 6,207,392, 6,423,551, 6,251,303, 6,306,610; 6,322,901, 6,319,426, 6,326,144, and 6,444,143, the entire contents of which are incorporated herein by reference for all purposes.

[0078] Detectable labels are commercially available or may be prepared by methods known to those skilled in the art. Detectable labels can be covalently attached to compounds using reactive functional groups that can be located at any suitable position. Methods for attaching detectable labels are known to those skilled in the art. When the reactive group is attached to an alkyl or substituted alkyl chain linked to an aryl nucleus, it may be located at the terminal position of the alkyl chain.

[0079] III. DETAILED DESCRIPTION OF EMBODIMENTS A. Treating Inflammatory Bowel Disease with Combination Therapy The present disclosure provides methods, compositions, and kits based on a combination therapy comprising a CCR9 inhibitor and an anti-α4β7 integrin antibody. The therapy is useful for treating IBD, such as Crohn's disease (CD) and ulcerative colitis (UC), in a subject. The present invention is based in part on the unexpected finding that a synergistic combination of a CCR9 inhibitor and an anti-α4β7 integrin antibody is effective in treating IBD. (1. Crohn's Disease)

[0080] The compositions, methods, and kits of the present invention can be used in patients with CD, including all forms of CD. The combination therapy of a CCR9 inhibitor and an anti-α4β7 integrin antibody can be administered in an effective amount to induce a clinical response or maintain clinical remission in a CD patient. In some embodiments, the combination therapy alleviates, relieves, or minimizes the severity of one or more symptoms of CD.

[0081] Symptoms of CD include diarrhea, fever, fatigue, abdominal pain or cramps, bloody stools, stomatitis, loss of appetite, weight loss, and perianal disease. Additional symptoms or features of CD can be assessed by endoscopic examination (e.g., esophagogastroduodenoscopy, colonoscopy, sigmoidoscopy, endoscopic retrograde cholangiopancreatography, endoscopic ultrasound, and balloon endoscopy) and histological examination of biopsy samples from the gastrointestinal tract. The severity of the disease can be classified as mild-moderate, moderate-severe, and severe / fulminant. Further description of CD can be found, for example, in Lichtenstein et al., Am J Gastroenterol, 2009, 104(2):2465-83.

[0082] The severity of CD and clinical response to combination therapy can be determined by clinical indices such as the Crohn's Disease Activity Index, or CDAI (Best et al., Gastroenterology, 1976, 70:439-44). This index is used to quantify symptoms in patients with CD. The CDAI can be used to define clinical response or remission of CD. The CDAI is composed of eight factors, which are added (summed) after adjusting for weighting factors or multipliers. The eight factors include frequency of liquid stools, abdominal pain, general health, extraintestinal symptoms, antidiarrheal medications, abdominal masses, hematocrit, and weight. Remission of Crohn's disease is generally defined as a decline or reduction in the CDAI to less than 150 points. Severity is generally defined as a value greater than 450 points. In one aspect, response of Crohn's disease patients to a particular drug is defined as a decline in the CDAI from baseline (0 weeks after treatment) by more than 70 points.

[0083] Clinical indicators such as CDAI can be used to determine whether the combination therapy described herein induces a clinical response or clinical remission in patients with Crohn's disease.In some embodiments, if the patient's CDAI score decreases by 70 points or more from baseline after combination therapy, the patient has a clinical response.If the patient's CDAI score decreases to less than 150 points at the end of the induction period of treatment, the patient is in clinical remission of CD.

[0084] (2.Ulcerative colitis) The compositions, methods, and kits of the present invention can be used in patients with UC, including all forms of UC. The combination therapy of a CCR9 inhibitor and an anti-α4β7 integrin antibody can be administered in an effective amount to induce a clinical response or maintain clinical remission in patients with UC. In some embodiments, the combination therapy relieves, alleviates, or minimizes the severity of one or more symptoms of UC.

[0085] Symptoms of UC include, but are not limited to, diarrhea, abdominal pain or cramps, rectal pain, rectal bleeding, urgency, inability to defecate, weight loss, fatigue, fever, and anemia. The severity of the disease can be classified as mild-moderate, moderate-severe, and severe / fulminant. See, e.g., Kornbluth et al., Am J Gastroenterol, 2004, 99(7):1371-85.

[0086] UC disease activity and response to treatment can be assessed by quantitative analysis using a composite index scoring system. In general, when assessing UC disease activity, clinicians consider at least four factors or variables: clinical symptoms, quality of life, endoscopic assessment, and histological assessment. For example, the Colitis Activity Index (CAI) is a quantitative measurement that incorporates colonic symptoms of inflammation in the colon, diarrhea, abdominal pain and cramps, and bloody stools based on colonoscopy. Standardized endoscopic scoring systems such as the UC Endoscopic Index of Severity (UCEIS) are useful for establishing a patient's disease index score. Other useful disease activity indices include the Mayo Clinic Score (see, e.g., Rutgeert et al., N Eng J Med, 2005, 353(23):2462-76) and the modified Mayo Disease Activity Index (MMDAI) (see, e.g., Schroeder et al., N Eng J Med, 1987, 317(26):1625-9). The four factors used in the Mayo Clinic scoring system include stool frequency, rectal bleeding, endoscopic findings, and the physician's global assessment of disease severity (e.g., daily abdominal discomfort and general well-being).

[0087] Compared to the Mayo Clinic Score, the MMDAI eliminates the "frailty" of an endoscopy score of 1. Thus, frailty is reflected by an endoscopy score of 2 or 3. The MMDAI assesses four subscores (stool frequency, rectal bleeding, endoscopic findings, and physician's global assessment), each of which ranges from 0 to 3, with a maximum total score of 12.

[0088] In some embodiments, a UC patient's clinical response to a combination therapy described herein corresponds to a reduction in the MMDAI score of 2 or more points from baseline and a reduction of 25% or more from baseline, and / or a reduction in the rectal bleeding subscore of 1 or more points. In other embodiments, a clinical response corresponds to a reduction in the Mayo Clinic Score of 3 or more points or a reduction of 30% from baseline.

[0089] Clinical remission in UC patients receiving combination therapy may correspond to an MMDAI subscale rectal bleeding score of 0 and a combined stool frequency and physician assessment score of ≤ 2. In other embodiments, clinical remission in UC patients refers to a Mayo Clinic Score of ≤ 2 with no subscores (stool frequency, rectal bleeding, endoscopic findings, and physician global assessment) of ≥ 2. (B. Combination therapy of CCR9 inhibitor and anti-α4β7 integrin antibody)

[0090] Provided herein are methods, compositions, and kits that utilize the synergistic effects of a CCR9 inhibitor and an anti-α4β7 integrin antibody to reduce inflammation in IBD patients. A combination therapy that includes both a CCR9 inhibitor and an anti-α4β7 integrin antibody is more effective in treating one or more symptoms of IBD than either compound / antibody alone.

[0091] (1. Chemokine receptor 9 (CCR9) inhibitors) The present invention provides compounds that modulate CCR9 activity. Specifically, the present invention provides compounds that have anti-inflammatory or immunomodulatory activity. The compounds of the present invention are believed to disrupt inappropriate T cell trafficking by specifically modulating or inhibiting chemokine receptor function. Chemokine receptors are integral membrane proteins that interact with extracellular ligands, such as chemokines, and mediate cellular responses to the ligands (e.g., chemotaxis, increased intracellular calcium ion concentration, etc.). Thus, modulation of chemokine receptor function (e.g., disruption of chemokine receptor-ligand interaction) can inhibit or reduce chemokine receptor-mediated responses, and can also treat or prevent chemokine receptor-mediated conditions or diseases.

[0092] Without being bound to any particular theory, it is believed that the compounds described herein interfere with the interaction between CCR9 and its ligand CCL25.For example, the compounds of the present invention act as potent CCR9 antagonists, and this antagonistic activity is further confirmed in animal studies on inflammation, one of the prominent pathologies related to CCR9.The compounds contemplated by the present invention include, but are not limited to, the exemplary compounds described herein and their salts.

[0093] For example, useful compounds act as potent CCR9 antagonists, and this antagonistic activity is further confirmed in animal studies of inflammation, one of the prominent pathologies of CCR9. Accordingly, the compounds described herein are useful in pharmaceutical compositions and methods for treating inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease).

[0094] In some embodiments, the CCR9 inhibitor (e.g., a small molecule CCR9 inhibitor of the disclosure) is a compound represented by formula (I) or a salt thereof: [ka]

[0095] (In the formula, R 1 is a substituted or unsubstituted C 2~8Alkyl, substituted or unsubstituted C 1~8 Alkoxy, substituted or unsubstituted C 1~8 Alkylamino, and substituted or unsubstituted C 3~10 heterocyclyl;

[0096] R 2 is H, F, Cl, or substituted or unsubstituted C 1~8 Is it alkoxy;

[0097] R 1 and R 2 form, together with the carbon atom to which they are attached, a non-aromatic carbocyclic or heterocyclic ring;

[0098] R 3 is H, substituted or unsubstituted C 1~8 Alkyl, substituted or unsubstituted C 1~8 alkoxy, or halo;

[0099] R 4 is H or F;

[0100] R 5 is H, F, Cl, or -CH3;

[0101] R 6 H, halo, -CN, -COR a , -CONH2, -NH2, substituted or unsubstituted C 1~8 Alkyl, substituted or unsubstituted C 1~8 Alkoxy or substituted or unsubstituted C 1~8 is an aminoalkyl;

[0102] R a is H or substituted or unsubstituted C 1~8 is alkyl;

[0103] Here, R 5 and R 6 may together form a carbocyclic ring;

[0104] L is a bond, -CH2-, or -CH(CH3)-;

[0105] A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , and A 8 are N, NO, and -CR 8 -, wherein A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , and A 8 of which 1-2 are N or NO;

[0106] Each R 8 is H, halo, -CN, -OH, oxo, substituted or unsubstituted C 1~8 Alkyl, substituted or unsubstituted C 1~8 Alkoxy, -NR 20 R 21 , independently selected from the group consisting of substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclyl; and

[0107] R 20 and R 21 each independently represents H or a substituted or unsubstituted C 1~8 (It is alkyl).

[0108] In some embodiments of formula (I), A 1 Or A 2 One of the is N or NO, and A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , and A 8 The remainder is -CR 8 -, where each R 8 are independently selected.

[0109] Depending on the aspect, A 2 , A 3 , A 4 , and A 5 Two of them are N or NO, and A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , and A 8 The remainder is -CR 8 -, where each R 8 are independently selected.

[0110] In an embodiment, the compound or composition of Formula (I) is represented by Formula (II) or a salt thereof: [ka]

[0111] (In the formula, R 1 is a substituted or unsubstituted C 2~8 Alkyl, substituted or unsubstituted C 1~8 Alkoxy, substituted or unsubstituted C 1~8 Alkylamino, and substituted or unsubstituted C 3~10 heterocyclyl;

[0112] R 2 is H, F, Cl, or substituted or unsubstituted C 1~8 Is it alkoxy;

[0113] R 1 and R 2 form, together with the carbon atom to which they are attached, a non-aromatic carbocyclic or heterocyclic ring;

[0114] R 3 is H, substituted or unsubstituted C 1~8 Alkyl, substituted or unsubstituted C 1~8 alkoxy, or halo;

[0115] R 4 is H or F;

[0116] R 5 is H, F, Cl, or -CH3;

[0117] R 6 H, halo, -CN, -COR a , -CONH2, -NH2, substituted or unsubstituted C 1~8 Aminoalkyl, substituted or unsubstituted C 1~8 Alkyl, or substituted or unsubstituted C 1~8 is alkoxy;

[0118] R a is H or substituted or unsubstituted C 1~8 is alkyl;

[0119] Here, R 5 and R 6 may together form a carbocyclic ring;

[0120] L is a bond, -CH2-, or -CH(CH3)-; and

[0121] Z is [ka] and N-oxides thereof;

[0122] wherein the Z group may be unsubstituted or may contain 1 to 3 independently selected substituents R 8 may be substituted with;

[0123] Each R 8 is H, halo, -CN, -OH, oxo, substituted or unsubstituted C 1~8 Alkyl, substituted or unsubstituted C 1~8 Alkoxy, -NR 20 R 21, independently selected from the group consisting of substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclyl; and

[0124] R 20 and R 21 each independently represents H or a substituted or unsubstituted C 1~8 (It is alkyl).

[0125] In some embodiments of Formula (II), Z is selected from the group consisting of substituted or unsubstituted quinolinyl, substituted or unsubstituted isoquinolinyl, substituted or unsubstituted 1,6-naphthyridinyl, substituted or unsubstituted cinnolinyl, substituted or unsubstituted phthalazinyl, and substituted or unsubstituted quinazolinyl.

[0126] In one aspect, the compound or composition of formula (I) described herein has formula (IIIa) or (IIIb) or a salt thereof: [ka]

[0127] (In the formula, R 1 is a substituted or unsubstituted C 2~8 Alkyl, substituted or unsubstituted C 1~8 Alkoxy, substituted or unsubstituted C 1~8 Alkylamino, and substituted or unsubstituted C 3~10 heterocyclyl;

[0128] R 2 is H, F, Cl, or substituted or unsubstituted C 1~8 Is it alkoxy;

[0129] R 1 and R 2 form, together with the carbon atom to which they are attached, a non-aromatic carbocyclic or heterocyclic ring;

[0130] R 3 is H, substituted or unsubstituted C 1~8Alkyl, substituted or unsubstituted C 1~8 alkoxy, or halo;

[0131] R 4 is H or F;

[0132] R 5 is H, F, Cl, or -CH3;

[0133] R 6 H, halo, -CN, -COR a , -CONH2, -NH2, substituted or unsubstituted C 1~8 Aminoalkyl, substituted or unsubstituted C 1~8 Alkyl, or substituted or unsubstituted C 1~8 is alkoxy;

[0134] R a is H or substituted or unsubstituted C 1~8 is alkyl;

[0135] Or, here, R 5 and R 6 form a carbocyclic ring together with the carbon atom to which they are attached;

[0136] Each R 8 is H, halo, -CN, -OH, oxo, substituted or unsubstituted C 1~8 Alkyl, substituted or unsubstituted C 1~8 Alkoxy, -NR 20 R 21 , independently selected from the group consisting of substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclyl;

[0137] R 20 and R 21 each independently represents H or a substituted or unsubstituted C 1~8 is alkyl; and

[0138] where n is 0, 1, 2, or 3).

[0139] In one embodiment of formula (IIIa) or (IIIb) or a salt thereof, R 1 is selected from the group consisting of -CH2CH3, -CH(CH3)2, -C(CH3)3, -C(CH3)2CH2CH3, -C(CH2CH2)CN, -C(OH)(CH3)2, -OCH3, -OCH2CH3, -OCH(CH3)2, -OC(CH3)3, -OCH2CH(CH3)2, -OCF3, and morpholino; R 2 is H, F, or Cl; R 1 and R 2 may together form -OC(CH3)2CH2- or -C(CH3)2CH2CH2-; R 3 is H, -CH3, or -OCH3; R 4 is H or F; R 5 is H;R 6 is H, -CH3, -CH2CH3, -CH(CH3)2, -C3H7, -CH2F, -CHF2, -CF2CH3, -CF3, -CH2OCH3, -CH2OH, -CH2CN, -CN, or -CONH2; and each R 8 is independently selected from the group consisting of H, F, Cl, Br, -CH3, -OH, -OCH3, -OCH2CH3, -NH2, -N(CH3)2, and -CN. 1 is -C(CH3)3.

[0140] In another embodiment of Formula (IIIa) or Formula (IIIb), R 2 is H or F; R 3 is H;R 4 is H; and R 6 is -CH3, -CH2F, -CHF2, or -CF3.

[0141] In one embodiment, the compounds and compositions of formula (IIIa) or (IIIb) or salts thereof are selected from the group consisting of: [ka] [ka]

[0142] In some embodiments, the CCR9 inhibitor (e.g., the small molecule CCR9 inhibitor compounds and compositions described herein) is selected from the group consisting of: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and its N-oxide.

[0143] In some embodiments, preferred substituents R 1 In formula (I, II, IIIa, IIIb), R 1 is a substituted or unsubstituted C 2~8 Alkyl, substituted or unsubstituted C 1~8 Alkoxy, substituted or unsubstituted C 1~8 Alkylamino, and substituted or unsubstituted C 3~10 R is selected from the group consisting of heterocyclyl. 1 When R is a substituted alkyl, the alkyl group is preferably substituted with halo or hydroxy. 1 When R is a substituted alkoxy, the alkoxy group is preferably substituted with halo. 1 is unsubstituted C 2~8 Alkyl(C 3~8 (including cycloalkyl), C2~8 Haloalkyl, C 1~8 Hydroxyalkyl, unsubstituted C 1~8 Alkoxy, C 1~8 Haloalkoxy, and C 1~8 Alkylamino is preferred; unsubstituted C 2~8 Alkyl, C 2~8 Haloalkyl, unsubstituted C 1~8 Alkoxy, and C 1~8 Alkylamino is more preferred; unsubstituted C 2~8 Alkyl, unsubstituted C 1~8 Alkoxy, and morpholino are more preferred; unsubstituted C 2~8 is more preferred; t-butyl is most preferred.

[0144] In some embodiments, preferred substituents R 6 In formula (I, II, IIIa, IIIb), R 6 H, halo, -CN, -COR a , -CONH2, -NH2, substituted or unsubstituted C 1~8 Alkyl, substituted or unsubstituted C 1~8 Alkoxy or substituted or unsubstituted C 1~8 R is an aminoalkyl. 6 When R is a substituted alkyl, the alkyl group is preferably substituted with halo, hydroxy, alkoxy, or cyano. 6 -CN, -CONH2, -NH2, unsubstituted C 1~8 Alkyl, unsubstituted C 1~8 Haloalkyl, and unsubstituted C 1~8 Alkoxy is preferred; unsubstituted C 1~8 Alkyl or unsubstituted C 1~8 Haloalkyl is more preferred; unsubstituted C 1~8 Alkyl is more preferred; methyl is most preferred.

[0145] In some embodiments, the small molecule CCR9 inhibitor compounds of the invention include: [ka] It is.

[0146] In one embodiment, the small molecule CCR9 inhibitor is versanon (Traficet-EN™) or CCX507.

[0147] Detailed descriptions of the CCR9 inhibitor compounds described herein and methods for their preparation are described, for example, in U.S. Pat. No. 8,916,601; U.S. Patent Application Publication Nos. 2013 / 0267492, 2013 / 0059893, 2012 / 0245138, 2012 / 0165303, 2011 / 0021523, 2010 / 0331302, 2010 / 0227902, 2010 / 0190762, 2010 / 0152186, 2010 / 0056509, 2009 / 0163498, and 2009 / 0005410, the entire disclosures of which are incorporated herein by reference for all purposes.

[0148] The compounds described herein can be synthesized using a variety of standard organic chemical transformations. Certain general reaction types that are commonly used in the synthesis of the target compounds of the present invention are summarized in the Examples. In particular, general procedures for the formation of sulfonamides and the formation of N-oxides of azaaryls are described and routinely used.

[0149] Although not intended to be exhaustive, representative organic synthesis transformations that can be used to prepare the compounds of the present invention are listed herein. These include standard functional group manipulations; reduction (such as nitro to amino); oxidation of functional groups such as alcohols and azaaryls; aryl substitution by IPSO or other mechanisms to introduce various groups such as nitriles, methyls, halogens, etc.; introduction and removal of protecting groups; Grignard formation and reaction with electrophiles; cross-coupling with metals (including but not limited to the Buchwald reaction, Suzuki reaction, Sonogashira reaction, etc.); halogenation and other aromatic electrophilic substitution reactions; formation of diazonium salts and reactions of these species; etherification; cyclocondensations leading to heteroaryl rings, dehydration, oxidation, and reduction; aryl metallation and transmetallation reactions followed by reaction of aryl metal species with electrophiles (such as acid chlorides or Weinreb amides); amidation; esterification; nucleophilic substitution reactions; alkylation; acylation; sulfonamide formation; chlorosulfonylation; ester hydrolysis and related hydrolysis.

[0150] Certain molecules claimed in this application may exist in different enantiomeric and diastereomeric forms, and all such variations of these compounds are included within the scope of the invention. 8 Where is OH and is ortho to the nitrogen, it is represented by the formula --N.dbd.C(OH)--, however the tautomer --NH--C(O)-- is also intended to be included within the scope of this formula.

[0151] The synthetic descriptions below include precursors obtained from commercial sources, including Aldrich Chemical Co., Acros Organics, Ryan Scientific Incorporated, Oakwood Products Incorporated, Lancaster Chemicals, Sigma Chemical Co., Lancaster Chemical Co., TCI-America, Alfa Aesar, Davos Chemicals, and GFS Chemicals.

[0152] 2. Pharmaceutical Formulations of CCR9 Inhibitors In another aspect, the disclosure provides compositions or formulations for modulating CCR9 activity. Generally, compositions or formulations for modulating chemokine receptor activity in a subject, such as a human or animal, include a compound described herein and a pharma- ceutically acceptable excipient or diluent.

[0153] As used herein, the term "composition" is intended to encompass a product containing the specified ingredients in the specified amounts, as well as any product resulting directly or indirectly from combining the specified ingredients in the specified amounts. "Pharmaceutically acceptable" means that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the patient.

[0154] The pharmaceutical composition for administering the compound of the present invention may be conveniently provided in unit dosage form and can be prepared by any method well known in the field of pharmacy.All methods include the step of combining active ingredient with carrier that constitutes one or more accessory ingredients.In general, pharmaceutical composition is prepared by uniformly and intimately combining active ingredient with liquid carrier or finely divided solid carrier or both, and then shaping the product into desired formulation as necessary.Pharmaceutical composition contains the desired active compound in an amount sufficient to obtain the desired effect on the process or condition of disease.

[0155] In some embodiments, the CCR9 inhibitor of the present disclosure is a pharmaceutical compound having a crystalline form. Examples of such crystalline forms of CCR9 inhibitors are described, for example, but not limited to, in U.S. Patent No. 9,133,124, the entire disclosure of which is incorporated herein by reference for all purposes.

[0156] Pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, such as tablets, troches, drops, aqueous or oily suspensions, dispersible powders or granules, emulsions and self-emulsifying formulations (such as those in U.S. Pat. No. 6,451,399), hard or soft capsules, or syrups or elixirs. Compositions intended for oral use can be prepared according to any method known in the art for producing pharmaceutical compositions. Such compositions may contain one or more agents selected from sweeteners, flavoring agents, coloring agents, and preservatives to provide a phytically elegant and palatable preparation. Tablets contain the active ingredient in admixture with other excipients that are non-toxic, pharma- ceutically acceptable, and suitable for the manufacture of tablets. These excipients may be, for example, inert diluents (such as cellulose, silicon dioxide, aluminum oxide, calcium carbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose, calcium phosphate, or sodium phosphate); granulating and disintegrating agents (such as corn starch or alginic acid); binding agents (such as PVP, cellulose, PEG, starch, gelatin, or acacia); and lubricants (such as magnesium stearate, stearic acid, or talc). The tablets may be uncoated or may be coated by enteric coating or other known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be used. Tablets may also be coated for sustained release by techniques described in U.S. Pat. Nos. 4,256,108, 4,166,452, and 4,265,874 to form osmotic therapeutic tablets.

[0157] Formulations for oral use may further be provided as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin, or olive oil). Emulsions may also be prepared with water-immiscible ingredients (e.g., oils) and stabilized with surfactants (e.g., mono-, diglycerides, PEG esters, etc.).

[0158] Aqueous suspensions contain the active ingredient in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia); dispersing or wetting agents may be natural phospholipids (e.g., lecithin), or condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearates), ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), ethylene oxide with partial esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitol monooleate), or ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene sorbitan monooleate). The aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0159] Oily suspensions can be formulated by suspending the active ingredient in a vegetable oil (for example, arachis oil, olive oil, sesame oil, or coconut oil) or in a mineral oil (such as liquid paraffin). Oily suspensions may contain a thickening agent (for example, beeswax, hard paraffin, or cetyl alcohol). Sweetening agents (such as those set forth above), and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

[0160] Dispersible powders or granules suitable for preparation of an aqueous suspension by adding water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Examples of suitable dispersing or wetting agents and suspending agents are those already mentioned above. Additional excipients (e.g. sweetening, flavoring, and coloring agents) may also be included.

[0161] The pharmaceutical compositions of the invention may be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil (e.g. olive oil or arachis oil) or a mineral oil (e.g. liquid paraffin), or a mixture thereof. Suitable emulsifying agents include natural gums (e.g. gum acacia or gum tragacanth), natural phospholipids (e.g. soybean lecithin), esters or partial esters derived from fatty acids and hexitol anhydrides (e.g. sorbitan monooleate), and condensation products of such partial esters with ethylene oxide (e.g. polyethylene sorbitan monooleate). The emulsions may further contain sweetening and flavoring agents.

[0162] Syrups and elixirs can be formulated with sweetening agents (e.g., glycerin, propylene glycol, sorbitol, or sucrose). Such formulations may further contain analgesics, preservatives, flavorings, and coloring agents. Oral solutions can be prepared in combination with, for example, cyclodextrin, PEG, and surfactants.

[0163] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. The suspension may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents as mentioned above. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. Sterile axed oils are also conventionally used as a solvent or suspending medium. Any mild, fixed oil, such as synthetic mono- or diglycerides, may be used for this purpose. Fatty acids, such as oleic acid, are also used in the preparation of injectables.

[0164] The compounds disclosed herein can also be administered in the form of suppositories for rectal administration of the drug. Such compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, thereby melting in the rectum and releasing the drug. Such materials are cocoa butter and polyethylene glycol. The compounds can also be administered by intraocular delivery with solutions or ointments. In addition, transdermal delivery of the compounds of the present invention can be achieved by iontophoretic patches and the like.

[0165] Topical use employs creams, ointments, jellies, solutions or suspensions containing the compounds of the invention. As used herein, topical use is also intended to include the use of mouthwashes and gargles.

[0166] The pharmaceutical compositions and methods of the present invention may further comprise other therapeutically active compounds, as described herein, such as those used to treat the conditions mentioned above.

[0167] (3. Anti-α4β7 integrin blocking antibody) Anti-α4β7 integrin antibodies suitable for use in treating inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis) include antibodies from any desired source that inhibit the binding of α4β7 integrin to any one of its ligands (mucosal addressin cell adhesion molecule (MAdCAM-1), fibronectin, vascular cell adhesion molecule (VCAM), etc.). Anti-α4β7 integrin antibodies may be human antibodies, mouse antibodies, rabbit antibodies, modified antibodies (chimeric antibodies, humanized antibodies, etc.), and antigen-binding fragments of antibodies (Fab fragments, Fv fragments, scFv fragments, Fab' fragments, and F(ab')2 fragments, etc.).

[0168] In some embodiments, the anti-α4β7 integrin antibody binds to an epitope in the α4 chain, an epitope in the β7 chain, or a composite epitope formed by the association of the α4 and β7 chains. Optionally, the anti-α4β7 integrin antibody binds to an epitope formed by the association of the α4 and β7 chains, and does not bind to an epitope in the α4 or β7 chain when the α4 and β7 chains are not associated or complexed.

[0169] Examples of anti-α4β7 integrin antibodies for use in the methods described herein include, but are not limited to, vedolizumab (ENYVIO®), natalizumab (Tysabri®), AMG181 (Amgen), and those described in, for example, International Patent Publication No. 2012 / 151248, U.S. Patent Nos. 7,147,851, 7,402,410, 8,444,981, 8,454,961, 8,454,962, 8,871,490, and U.S. Patent Publication No. 2015 / 0086563 (the disclosures of which are incorporated herein by reference in their entirety for all purposes). Other useful anti-α4β7 integrin antibodies include bioequivalents, biosimilars, and biobetters of any of the anti-α4β7 integrin antibodies described herein.

[0170] In some embodiments, the anti-α4β7 integrin antibodies of the present disclosure are antibodies having an amino acid sequence that has at least 70%, at least 80%, at least 90%, at least 95% or more sequence identity to a reference anti-α4β7 integrin antibody (such as vedolizumab, AMG181, or other anti-α4β7 integrin antibodies known to those of skill in the art). In some cases, antibody variants have one or more amino acid substitutions, deletions, and / or additions at specific amino acid positions of the reference antibody, but retain antigen binding activity.

[0171] Those skilled in the art will recognize that "percent sequence identity" can be determined by comparing two optimally aligned sequences over a sequence comparison window or designated region, where the portion of the polypeptide sequence within the comparison window may have additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) when the two sequences are optimally aligned. The percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue is present in both sequences to determine the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to determine the percentage of sequence identity. Percent sequence identity can be measured using the sequence comparison algorithms BLAST or BLAST 2.0 using default parameters, or by manual alignment and visual inspection.

[0172] Antibodies and their fragments, variants, and derivatives can be produced by a variety of standard methods recognized by those skilled in the art. See, for example, Harlow, E. and Lane DP. Antibodies: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1988. Antigen-binding fragments, such as Fab and F(ab')2 fragments, can be produced by genetic engineering. Procedures for producing chimeric and further engineered monoclonal antibodies include those described in Riechmann et al., Nature, 1988,332:323; Liu et al., Proc. Nat. Acad. Sci. USA, 1987, 84:3439; Larrick et al., Bio / Technology, 1989, 7:934; and Winter et al., TIPS, 1993, 14:139. Examples of methods for producing and using transgenic animals for the production of human or partially human antibodies are described, for example, in Davis et al., 2003, Production of human antibodies from transgenic mice, Lo, ed. Antibody Engineering Methods and Protocols, Humana Press, NJ:191-200.

[0173] 4. Pharmaceutical preparations of anti-α4β7 integrin antibodies Provided herein is a formulation of an anti-α4β7 integrin antibody that can stabilize the antibody, reduce the formation of antibody aggregates, delay the degradation of the antibody, and / or minimize the immunogenicity of the antibody. The formulation can include an antioxidant or chelating agent, at least one free amino acid, a surfactant, a non-reducing sugar, and / or a buffering agent.

[0174] The antioxidant or chelating agent may be citrate, ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), dimercaprol, diethylenetriaminepentaacetic acid, or N,N-bis(carboxymethyl)glycine, with citrate or EDTA being preferred.The free amino acids may be histidine, alanine, arginine, glycine, glutamic acid, and combinations thereof. The surfactants include polysorbate 20; polysorbate 80; TRITON® (t-octylphenoxypolyethoxyethanol) (nonionic surfactant); sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; linoleyl betaine, myristyl betaine, or cetyl betaine; lauramidopropyl betaine, cocamidopropyl betaine, linoleyl betaine, or linoleyl betaine. The surfactant may be lauramidopropyl betaine, myristamidopropyl betaine, palmamidopropyl betaine, or isostearamidopropyl betaine (e.g., lauramidopropyl betaine); myristamidopropyl dimethylamine, palmamidopropyl dimethylamine, or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; sorbitan monopalmitate; polyethyl glycol (PEG), polypropylene glycol (PPG), and copolymers of polyoxyethylene and polyoxypropylene glycol, with polysorbate 80 being preferred.

[0175] The buffering agent may be a buffering agent capable of adjusting the pH value of the formulation to about 5.0 to about 7.5, about 5.5 to about 7.5, about 6.0 to about 7.0, or about 6.3 to about 6.5. Examples of buffering agents include, but are not limited to, acetate, succinate, gluconate, histidine, citrate, phosphate, maleate, cacodylate, 2-[N-morpholino]ethanesulfonic acid (MES), bis(2-hydroxyethyl)iminotris[hydroxymethyl]methane (Bis-Tris), N-[2-acetamido]-2-iminodiacetic acid (ADA), glycylglycine, and other organic acid buffers, with histidine or citrate being preferred.

[0176] In some embodiments, the anti-α4β7 integrin antibody is a lyophilized formulation (e.g., in a dried form). In some cases, the lyophilized formulation comprises the anti-α4β7 integrin antibody and one or more excipients (such as a non-reducing sugar, a buffer, a free amino acid, and / or a surfactant).

[0177] In some cases, the lyophilized formulation contains at least about 50 mg, at least about 60 mg, at least about 70 mg, at least about 80 mg, at least about 90 mg, at least about 100 mg, at least about 120 mg, at least about 140 mg, at least about 180 mg, at least about 200 mg, at least about 220 mg, at least about 240 mg, at least about 280 mg, at least about 300 mg, at least about 400 mg, at least about 500 mg, at least about 600 mg, at least about 700 mg, at least about 800 mg, at least about 900 mg of an anti-α4β7 integrin antibody. In some cases, the lyophilized formulation is stored in a single vial as a single dose.

[0178] In some embodiments, the anti-α4β7 integrin antibody is in a liquid formulation. Such a formulation may include an anti-α4β7 integrin antibody, a buffer, a non-reducing sugar, and / or a free amino acid.

[0179] The amount of antibody contained in the liquid formulation may be at least about 25 mg / ml to about 200 mg / ml (e.g., 25 mg / ml to about 200 mg / ml, 25 mg / ml to about 150 mg / ml, 25 mg / ml to about 100 mg / ml, 50 mg / ml to about 200 mg / ml, 50 mg / ml to about 150 mg / ml, 50 mg / ml to about 100 mg / ml, 100 mg / ml to about 200 mg / ml, or 150 mg / ml to about 200 mg / ml) of anti-α4β7 integrin antibody.

[0180] The non-reducing sugar may be, but is not limited to, mannitol, sorbital, sucrose, trehalose, raffinose, stachyose, melezitose, dextran, maltitol, lactitol, isomaltulose, Palatinit®, and combinations thereof. In some embodiments, the ratio of non-reducing sugar to anti-α4β7 integrin antibody is at least 400:1 (mol:mol), at least 400:1 (mol:mol), at least 400:1 (mol:mol), at least 600:1 (mol:mol), at least 625:1 (mol:mol), at least 650:1 (mol:mol), at least 700:1 (mol:mol), at least 750:1 (mol:mol), at least 800:1 (mol:mol), at least 1000: 1 (mol:mol), at least 1100:1 (mol:mol), at least 1200:1 (mol:mol), at least 1300:1 (mol:mol), at least 1400:1 (mol:mol), at least 1500:1 (mol:mol), at least 1600:1 (mol:mol), at least 1700:1 (mol:mol), at least 1800:1 (mol:mol), at least 1900:1 (mol:mol), or at least 2000:1 (mol:mol).

[0181] Exemplary formulations of anti-α4β7 integrin antibodies include, but are not limited to, those described in, for example, U.S. Patent Application Publication Nos. 2012 / 0282249, 2014 / 0377251, and 2014 / 0341885, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0182] C. Methods of Administration of Combination Therapy In another aspect, the present disclosure provides a combination therapy for the treatment of IBD (e.g., CD and UC). The combination therapy comprises a therapeutically effective amount of a CCR9 inhibitor and a therapeutically effective amount of an anti-α4β7 integrin blocking antibody. The combination of therapeutic agents can act synergistically to treat or prevent various disorders. Using this method, therapeutic efficacy can be achieved with lower doses of each agent, thereby reducing the potential for adverse side effects.

[0183] The term "therapeutically effective amount" refers to an amount of a subject compound that elicits the biological or medical response in a cell, tissue, system, or animal (such as a human) that is desired by a researcher, veterinarian, physician, or other treatment provider.

[0184] The compounds, antibodies, and formulations of the present disclosure can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, intravenous vena cava (ICV), intracapsular injection or infusion, subcutaneous injection, or placement), inhalation, intranasal, intravaginal, rectal, sublingual, or topical routes of administration, depending on the disease state and patient condition. Additionally, these compounds and antibodies, alone or in combination, can be formulated into suitable dosage unit formulations containing conventional non-toxic pharmacologic carriers, adjuvants, and solvents appropriate for each administration route. The present disclosure further contemplates administering the compounds and antibodies of the present disclosure in sustained release formulations.

[0185] For the treatment of IBD (such as Crohn's disease and UC), suitable dosage levels of CCR9 inhibitors are generally about 0.001-100 mg / kg (of patient body weight) per day, and can be administered in a single dose or multiple doses. Dosage levels are preferably about 0.01-50 mg / kg per day; more preferably about 0.05-10 mg / kg per day. Suitable doses may be about 0.01-50 mg / kg per day, about 0.05-10 mg / kg per day, or about 0.1-5 mg / kg per day. Within this range, dosages may be 0.005-0.05 mg / kg per day, 0.05-0.5 mg / kg per day, 0.5-5.0 mg / kg per day, or 5.0-50 mg / kg per day.

[0186] For oral administration, the CCR9 inhibitor is preferably provided in the form of a tablet containing 1.0 to 1000 milligrams of active ingredient (particularly 1.0 mg, 5.0 mg, 10.0 mg, 15.0 mg, 20.0 mg, 25.0 mg, 50.0 mg, 75.0 mg, 100.0 mg, 150.0 mg, 200.0 mg, 250.0 mg, 300.0 mg, 400.0 mg, 500.0 mg, 600.0 mg, 750.0 mg, 800.0 mg, 900.0 mg, and 1000.0 mg), so that the dosage for the patient being treated can be adjusted symptomatically.

[0187] The CCR9 inhibitor may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day.

[0188] In the treatment of IBD (such as Crohn's disease and UC), an appropriate dose level of the anti-α4β7 integrin antibody provides an amount of the antibody or a formulation thereof effective to induce remission of IBD in a human patient. In some embodiments, a therapeutically effective amount of the anti-α4β7 integrin antibody is an amount sufficient to provide a mean serum trough concentration of the anti-α4β7 integrin antibody of about 5 μg / ml to about 60 μg / ml (e.g., about 5 μg / ml to about 60 μg / ml, about 10 μg / ml to about 50 μg / ml, about 15 μg / ml to about 45 μg / ml, about 20 μg / ml to about 30 μg / ml, about 25 μg / ml to about 35 μg / ml, or about 30 μg / ml to about 60 μg / ml) at the end of the induction phase of treatment.

[0189] Suitable dosages of the antibody can be administered from about 0.1 mg / kg, about 0.3 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or about 10 mg / kg.

[0190] In some embodiments, the total dosage is about 6 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 650 mg, or more.

[0191] In some embodiments, the induction period is at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, or at least about 10 weeks.

[0192] The treatment regimen during the induction phase may include administration of a high dose, a high frequency, or a combination of high dose and high frequency administration of the anti-α4β7 integrin antibody or formulation thereof. In some cases, a dose is administered daily, every other day, every second day, every third day, once a week, once every 10 days, once every two weeks, once every three weeks, or once a month during the induction phase.

[0193] In some embodiments, the induction phase is administered once at the beginning of treatment (day 0 of treatment) and once about 2 weeks after the start of treatment. The induction phase may last 6 weeks. In other embodiments, the induction phase lasts 6 weeks, with multiple induction doses administered in the first 2 weeks. For example, if a human patient has severe IBD or is unresponsive to anti-TNFα treatment, the induction phase will last longer than in a patient with mild to moderate IBD.

[0194] Additionally, in the treatment of IBD, an appropriate dose level of the anti-α4β7 integrin antibody provides an amount of the antibody or formulation thereof effective to maintain remission of IBD in a human patient. Thus, during the maintenance phase of treatment, an amount of the anti-α4β7 integrin antibody effective for treatment is an amount that maintains a steady-state average serum trough concentration of the anti-α4β7 integrin antibody during the maintenance phase of treatment within a range of about 1 μg / ml to about 25 μg / ml (e.g., about 1 μg / ml to about 25 μg / ml, about 1 μg / ml to about 20 μg / ml, about 1 μg / ml to about 15 μg / ml, about 1 μg / ml to about 10 μg / ml, about 1 μg / ml to about 5 μg / ml, about 5 μg / ml to about 2 ... The amount is sufficient to achieve a steady-state mean serum trough concentration at the end of the induction phase of the anti-α4β7 integrin antibody of about 20 μg / ml, about 5 μg / ml to about 15 μg / ml, about 5 μg / ml to about 10 μg / ml, about 15 μg / ml to about 25 μg / ml, about 15 μg / ml to about 20 μg / ml, about 10 μg / ml to about 25 μg / ml, about 10 μg / ml to about 20 μg / ml, about 10 μg / ml to about 15 μg / ml, or about 20 μg / ml to about 25 μg / ml.

[0195] The dosage during the maintenance phase can be administered once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks. In some embodiments during the maintenance phase, the same dosage is administered during the maintenance phase. In other embodiments during the maintenance phase, one or more different dosages are administered during the maintenance phase. Also, the frequency of administration may increase depending on the course of the disease.

[0196] The anti-α4β7 integrin antibody or a formulation thereof can be administered by injection (e.g., intravenous, intramuscular, subcutaneous, intraarterial, intraperitoneal, intravitreal, etc.). If the formulation is in a solid or lyophilized form, the method of administering the antibody can include reconstituting the dry formulation into a liquid formulation. In some embodiments, the antibody or a formulation thereof can be administered locally (e.g., in a patch, cream, spray, or suppository). In other embodiments, the local administration route includes intranasal, inhalation, or transdermal administration.

[0197] However, it will be appreciated that the specific dosage and frequency of administration for any particular patient may vary and will depend upon a variety of factors, such as the activity of the specific compound used, the metabolic stability and length of action of that compound, age, body weight, genetics, general health, sex, diet, mode and duration of administration, excretion rate, drug combination, the severity of the particular condition, the host being treated, and the like.

[0198] The weight ratio of the CCR9 inhibitor described herein to the anti-α4β7 integrin antibody of the present disclosure may vary and depends on the effective dose of each component. In general, an effective dose of each component is used. Thus, for example, when a CCR9 inhibitor is used in combination with an anti-α4β7 integrin antibody, the weight ratio of the CCR9 inhibitor to the anti-α4β7 integrin antibody generally ranges from about 1000:1 to about 1:1000, with about 200:1 to about 1:200 being preferred.

[0199] Combination therapy includes co-administration of a CCR9 inhibitor and an anti-α4β7 integrin antibody, sequential administration of a CCR9 inhibitor and an anti-α4β7 integrin antibody, administration of a composition comprising a CCR9 inhibitor and an anti-α4β7 integrin antibody, or simultaneous administration of separate compositions (one composition containing a CCR9 inhibitor and another composition containing an anti-α4β7 integrin antibody).

[0200] Co-administration includes administration of the CCR9 inhibitor of the present invention within 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 20 hours, or 24 hours of the anti-α4β7 integrin antibody of the present invention. Co-administration further includes administration simultaneously, approximately simultaneously (e.g., within about 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 30 minutes of each other), or sequentially in any order. Additionally, the CCR9 inhibitor and the anti-α4β7 integrin antibody can each be administered once or twice, three times, or more times a day to obtain a preferred daily dosage level.

[0201] The combination therapy can be administered during the induction or maintenance phase of the treatment regimen. During the induction phase, the combination therapy can be administered in an amount effective to induce immune tolerance to the therapeutic antibodies, induce a clinical response, and / or improve one or more symptoms of IBD. Also, if there is a recurrence of one or more symptoms of IBD during the maintenance phase, or a relapse from a remission phase of the disease, an amount equivalent to the treatment during the induction phase can be administered to the patient. During the maintenance phase, the combination therapy can be administered in an amount effective to continue the response achieved during the induction treatment and / or to prevent the recurrence or relapse of symptoms of IBD.

[0202] In some embodiments, one or more additional active ingredients, such as an anti-inflammatory compound (e.g., sulfasalazine, azathioprine, 6-mercaptopurine, 5-aminosalicylic acid-containing anti-inflammatory agents, nonsteroidal anti-inflammatory compounds, and steroidal anti-inflammatory compounds); antibiotics commonly administered for the management of IBD (e.g., ciprofloxacin and metronidazole); or another biologic agent (e.g., a TNFα antagonist), can be administered in conjunction with the combination therapies disclosed herein.

[0203] (D.Kit) In some aspects, provided herein is a kit useful for treating a disease or disorder characterized by inflammation of the gastrointestinal tract (e.g., IBD, such as CD, UC, indeterminate colitis, etc.) comprising a CCR9 inhibitor and an anti-α4β7 integrin antibody disclosed herein. The kit may include a pharmaceutical composition containing a CCR9 inhibitor compound (e.g., a small molecule CCR9 inhibitor) and a pharmaceutical composition containing an anti-α4β7 integrin antibody. In some embodiments, the CCR9 inhibitor compound is Versanon (Traficet-EN™) or CCX507. In some embodiments, the anti-α4β7 integrin antibody is vedolizumab. In some cases, the kit includes written materials (e.g., instructions for use of the compound, antibody, or pharmaceutical composition thereof). Without limitation, the kit may include buffers, diluents, filters, needles, syringes, and enclosed inserts containing instructions for practicing any of the methods disclosed herein. EXAMPLES

[0204] IV. EXAMPLES The following examples are provided to illustrate, but not to limit, the invention of this application.

[0205] (Example 1: Treatment of inflammatory bowel disease with combined therapy of CCR9 inhibitor and anti-α4β7 integrin antibody) (A. Introduction) Homing of circulating cells to various tissues is a highly coordinated process involving specific chemokine receptors and cell adhesion molecules. Trafficking of cells to the intestine requires chemotaxis to the chemokine known as CCL25, which involves the chemokine receptor CCR9. Activation of CCR9 by CCL25 also leads to high affinity binding of the cell surface α4β7 integrin to MAdCAM-expressing intestinal capillary endothelium, resulting in tight retention and extravasation into the intestinal tissue.

[0206] Analysis of human colon biopsy samples from patients with colonic Crohn's disease showed strong positive correlations between gene expression levels of CCR9 and TNF-α, as well as α4 and β7 integrins. These results indicate that regulation of genes involved in intestinal inflammation is strongly associated with and tightly regulated in the colon. Vedolizumab, a humanized antibody against α4β7 integrin, was recently approved to treat patients with moderate to severe ulcerative colitis and Crohn's disease. However, the effect of vedolizumab in combination with antagonists of the chemokine receptor CCR9, which is involved in homing to the intestine, has not been investigated.

[0207] (B. Method) Adhesion assays were performed as follows: Human lymphocytes were isolated from total PBMCs and activated with α-CD3ε and α-CD28 (1 μg / mL; R&D Systems) in the presence of 1 μM retinoic acid (Sigma) and 1 ng / mL human IL12 (R&D Systems). In addition, ex vivo activated T cells were expanded in the presence of 1 μM retinoic acid (RA) and 1 ng / mL human IL12 for 5 days. Expanded cells were stained with APC-conjugated anti-CCR9 antibody (cat. no. 248621; R&D Systems) and conjugated anti-α4β7 integrin antibody (Act-1). Stained cells were analyzed by flow cytometry. Expression of CCR9 and α4β7 integrin on expanded T cells is shown as a two-parameter scatter plot in Figure 1A.

[0208] In vitro activated T cells were added to 96-well plates coated overnight with 0.3 μg / mL MAdCAM-1-Fc fusion protein (R&D Systems) in the following conditions: DMSO alone, 500 ng human CCL25 (hCCL25; a ligand for CCR9), 1 μM CCX507 (a small molecule CCR9 inhibitor), or 500 ng hCCL25 and 1 μM CCX507. Adherent cells were quantified using a CyQUANT® Cell Proliferation Assay (Thermo Fisher). Results are shown in Figure 1B. Data indicate that CCX507 restricts binding of RA-differentiated human T cells to MAdCAM-1, which can bind to α4β7 integrin and promote lymphocyte trafficking.

[0209] In vivo pharmacodynamic analysis was performed as follows. Figure 2A shows the adoptive T cell transfer model used in the experiment. Details of the method are described, for example, in Tubo et al., PLOS One, 2012, 7(11):e50498. Briefly, CD8 positive T cells were isolated from T cell receptor (TCR) transgenic donor mice (OT-1) (B6.CD45.2). White blood cells were isolated from the spleen and lymph nodes. The isolated CD8 positive T cells were adoptively transferred into wild-type mice (B6.CD45.1). 1 × 10 6 pieces~1×10 7 Naive CD8 positive T cells were injected intraperitoneally into recipient mice.

[0210] 24 hours after transfer, recipient mice CD45.1 were orally challenged with cholera toxin alone or cholera toxin plus ovalbumin. Mice challenged with cholera toxin were used as a mouse model of intestinal inflammation. After 96 hours, leukocytes were isolated from the spleen, lymph nodes, small intestine, colon, blood, and liver. After 96 hours of challenge, the number of donor-derived CD8+ T cells in the small intestine was determined by flow cytometry. Details of the method are described, for example, in Tubo et al., PLOS One, 2012, 7(11):e50498.

[0211] Furthermore, mice exposed to cholera toxin were administered 5-30 mg / kg CCX507 or a certain dose of anti-α4β7 integrin antibody. The number of donor (OT-1)-derived CD8-positive intraepithelial lymphocytes (IEL) was counted. The results are shown in Figure 2B.

[0212] The QuantiGene® Plex Assay (Affymetrix) was performed as follows: Paired ileal and colonic biopsies from Crohn's disease patients were snap frozen in liquid nitrogen. Samples were homogenized according to the manufacturer's protocol. Gene expression was analyzed using a custom 37-plex panel (Affymetrix) and expression was normalized to the housekeeping gene cyclophilin. Figure 3 shows a comparison of CCR9 expression levels with selected genes.

[0213] A mouse model of piroxicam-promoted colitis (chemically induced colitis) was generated as follows. Seven-week-old Mdr1a - / - Mice and FVB (control) strain were fed 200 ppm piroxicam (Sigma) mixed with powdered diet in powdered diet feeding jars (Dyets, Inc.) for 10–12 days. Regular diet was placed on a wire mesh lid during the experimental period. Each mouse received 100 μg of anti-α4β7 integrin blocking antibody (DATK32) every other day (q2d) by intraperitoneal injection (IP). During the first 11 days when piroxicam was added to the powdered diet, each mouse received 300 μg of anti-TNFα antibody (XT3.11) every other day (qd) by intraperitoneal injection (IP). After piroxicam was removed, each mouse received 300 μg of anti-TNFα antibody (XT3.11) every other day (q2d) by intraperitoneal injection (IP). During the study, 30 mg / kg of CCX507 (a small molecule CCR9 inhibitor) was administered subcutaneously (SC) daily (qd). Mice used in the study were observed and treated according to the Institutional Animal Care and Use Committee (IACUC) approved procedures. Figure 4A shows the experimental design.

[0214] Figure 4B shows representative images of the colons of mice given vehicle control (1% HPMC), rat IgG2A isotype control, CCX507 (a small molecule CCR9 inhibitor molecule) alone, anti-α4β7 integrin blocking antibody alone, or CCX507 together with anti-α4β7 integrin blocking antibody. Figures 4C and 4D show quantified colon-to-body weight ratios. Figure 4C shows the combination of CCX507 and anti-α4β7 integrin blocking antibody, and Figure 4D shows the combination of CCX507 and anti-TNFα blocking antibody. Statistical analysis was performed with GraphPad Prism® (GraphPad Software). p values ​​are represented as follows: * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

[0215] At necropsy, formalin-fixed colon tissues were histopathologically evaluated in a blinded manner by an independent pathologist. Histologic scores for inflammation, loss of glandular epithelium, and erosions were added to obtain a total histologic score, which was based on the percentage of affected tissue in each section (a total of six sections were cut per mouse).

[0216] Figure 5A shows representative images of both the proximal and distal colons of individual mice, representing the mean histopathological score for each group. The combination of CCX507 and anti-α4β7 antibody is shown on the right.

[0217] The sum of histological scores for all mice tested is shown in Figure 5B. Statistical analysis was performed with GraphPad Prism®. p values ​​are represented as follows: * indicates p<0.05, **** indicates p<0.0001.

[0218] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will recognize that certain changes and modifications may be practiced within the scope of the appended claims, and each reference cited herein is hereby incorporated by reference in its entirety as if each reference was individually incorporated by reference.

Claims

1. 1. A composition for treating or reducing the incidence of inflammatory bowel disease in a mammal comprising a chemokine receptor CCR9 inhibitor, said composition being administered in combination with an anti-α4β7 integrin blocking antibody, wherein said chemokine receptor CCR9 inhibitor and the anti-α4β7 integrin blocking antibody have a synergistic effect.

2. 2. The composition of claim 1, wherein the anti-α4β7 integrin blocking antibody is vedolizumab.

3. The chemokine receptor CCR9 inhibitor is the following compound: 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof, or an N-oxide thereof.

4. The composition of claim 1, wherein the chemokine receptor CCR9 inhibitor and the anti-α4β7 integrin blocking antibody are administered in synergistic amounts.

5. 2. The composition of claim 1, wherein the inflammatory bowel disease is Crohn's disease (CD) or ulcerative colitis (UC).

6. The composition of claim 1 , wherein the composition and the anti-α4β7 integrin blocking antibody are administered in a combined formulation.

7. The composition of claim 1 , wherein the composition and the anti-α4β7 integrin blocking antibody are administered sequentially.

8. The composition of claim 1 , wherein the composition is administered prior to the anti-α4β7 integrin blocking antibody.

9. The composition of claim 1 , wherein the composition is administered after administration of the anti-α4β7 integrin blocking antibody.

10. The composition comprises: i) a TNFα antagonist; and ii) A pharma- ceutically acceptable carrier or excipient The composition of claim 1 further comprising:

11. A composition for treating or reducing the incidence of inflammatory bowel disease in a mammal comprising a chemokine receptor CCR9 inhibitor, said composition being administered in combination with an anti-TNFα blocking antibody, wherein said chemokine receptor CCR9 inhibitor and the anti-TNFα blocking antibody have a synergistic effect.

12. The composition of claim 11 , wherein the anti-TNFα blocking antibody is infliximab or adalimumab.

13. The chemokine receptor CCR9 inhibitor is the following compound: 【Chemistry 2】 or a pharma- ceutically acceptable salt thereof, or an N-oxide thereof.

14. The composition of claim 11 , wherein the chemokine receptor CCR9 inhibitor and the anti-TNFα blocking antibody are administered in synergistic amounts.

15. The composition of claim 11, wherein the inflammatory bowel disease is Crohn's disease (CD) or ulcerative colitis (UC).

16. The composition of claim 11 , wherein the composition and the anti-TNFα blocking antibody are administered in a combined formulation.

17. The method of claim 11 , wherein the composition and the anti-TNFα blocking antibody are administered sequentially.

18. The composition of claim 11 , wherein the composition is administered prior to the anti-TNFα blocking antibody.

19. The composition of claim 11 , wherein the composition is administered after administration of the anti-TNFα blocking antibody.

20. The composition comprises: i) a TNFα antagonist; and ii) A pharma- ceutically acceptable carrier or excipient The composition of claim 11 further comprising:

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