Methods for treating inflammatory bowel disease using alpha4beta7 integrin antagonists

Abstract

The present invention relates to methods of treating inflammatory bowel disease comprising the use of engineered peptides that bind to alpha4beta7 integrin (e.g., peptide monomers and dimers that include a disulfide bond or a thioether intramolecular bond). In one aspect, the present disclosure provides a method of treating inflammatory bowel disease (IBD) in a subject in need thereof, comprising administering to the subject an alpha4beta7 integrin antagonist, wherein the antagonist is administered orally to the patient at a dose of about 100 mg to about 500 mg once or twice daily, wherein the antagonist is a peptide dimer compound comprising two peptides or a pharmaceutically acceptable salt thereof.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 959,854, filed January 10, 2020, which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This application was filed electronically via EFS-Web and contains a sequence list submitted electronically in .txt format. The .txt file contains a sequence list named "PRTH_052_01WO_ST25.txt" created on January 8, 2021, and is approximately 7 kilobytes in size. The sequence list contained in this .txt file is part of the specification and is incorporated herein by reference in its entirety. Technical Field

[0005] This disclosure relates to a method of treating inflammatory bowel disease using engineered peptides (e.g., peptide monomers and dimers comprising disulfide bonds or thioether intramolecular bonds) that bind to α4β7 integrin. Background Technology

[0006] Integrins are non-covalently associated α / β heterodimeric cell surface receptors involved in numerous cellular processes ranging from cell adhesion and migration to gene regulation (Dubree et al., Selective α4β7 Integrin Antagonist and Their Potential as Anti-inflammatory Agents, *Journal of Medicinal Chemistry*, 2002, 45, 3451-3457). Differential expression of integrins can modulate cell adhesion properties, allowing different leukocyte populations to be recruited to specific organs in response to different inflammatory signals. If left unchecked, integrin-mediated adhesion processes can lead to chronic inflammation and autoimmune diseases.

[0007] α4 integrins α4β1 and α4β7 play important roles in lymphocyte migration throughout the gastrointestinal tract. These integrins are expressed on most leukocytes containing B and T lymphocytes, and mediate cell adhesion at these leukocytes by binding to their respective major ligands, vascular cell adhesion molecule (VCAM) and mucosal addressing cell adhesion molecule 1 (MAdCAM1). The binding specificity of these proteins differs, as VCAM binds to α4β1 and to a lesser extent to α4β7, while MAdCAM1 is highly specific for α4β7. In addition to pairing with the α4 subunit, the β7 subunit also forms a heterodimeric complex with the αE subunit to form α4β7, which is primarily expressed on intraepithelial lymphocytes (IELs) of the intestine, lung, and genitourinary tract. α4β7 is also expressed on dendritic cells in the intestine. The α4β7 heterodimer binds to E-cadherin on epithelial cells. IEL cells are thought to provide a mechanism for immune surveillance within the epithelial compartments. Therefore, blocking both α4β7 and α4β7 together may be a useful approach for treating inflammatory symptoms in the gut.

[0008] Inhibitors of specific integrin-ligand interactions have proven effective as anti-inflammatory agents for the treatment of various autoimmune diseases. For example, monoclonal antibodies exhibiting high binding affinity for α4β7 have shown therapeutic benefits against gastrointestinal autoinflammatory / autoimmune diseases such as Crohn's disease and ulcerative colitis (ID). However, these therapies interfere with α4β1 integrin-ligand interactions, thereby producing dangerous side effects for patients. Therapies using small molecule antagonists have shown similar side effects in animal models, thus hindering further development of these technologies. Recently engineered peptides exhibiting high potency and stability, as well as high specificity for α4β7 integrins, have proven effective in the treatment of various immune disorders, including inflammatory bowel disease.

[0009] However, there is a need in the art for alternative methods of treating inflammatory conditions using α4β7 antagonists and other agents. Such methods are disclosed herein. Summary of the Invention

[0010] This disclosure provides compositions and methods for treating various diseases and symptoms associated with α4β7 integrin signaling.

[0011] On one hand, this disclosure provides a method for treating inflammatory bowel disease (IBD) in a subject in need, the method comprising administering an α4β7 integrin antagonist to the subject orally at a dose of about 100 mg to about 500 mg once or twice daily, wherein the antagonist is a peptide dimer compound comprising two peptides or a pharmaceutically acceptable salt thereof; wherein each of the two peptides comprises or consists of any of the following sequences (optionally having an N-terminal Ac):

[0012] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Glu)-(D-Lys)-OH (SEQ ID NO: 1);

[0013] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Gly-(D-Lys)-OH (SEQ ID NO: 2);

[0014] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Pro-(D-Lys)-OH (SEQ ID NO: 3);

[0015] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Pro)-(D-Lys)-OH (SEQ ID NO: 4);

[0016] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-OH (SEQ ID NO: 5);

[0017] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-NH2 (SEQ ID NO: 5);

[0018] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-OH (SEQ ID NO: 6);

[0019] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-NH2 (SEQ ID NO: 6);

[0020] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Pro-(D-Lys)-OH (SEQ ID NO: 7);

[0021] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Pro-(D-Lys)-NH2 (SEQ ID NO: 7);

[0022] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Pro)-(D-Lys)-OH (SEQ ID NO: 8); or

[0023] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Pro)-(D-Lys)-NH2 (SEQ ID NO: 8);

[0024] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a Pen or a disulfide bond between two Pens, wherein the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and wherein the linker moiety is diethylene glycol (DIG).

[0025] In one embodiment, each of the two peptides comprises the following sequence:

[0026] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Glu)-(D-Lys)-OH (SEQ ID NO: 1),

[0027] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0028] In one embodiment, each of the two peptides comprises the following sequence:

[0029] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Gly-(D-Lys)-OH (SEQ ID NO: 2);

[0030] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0031] In one embodiment, each of the two peptides comprises the following sequence:

[0032] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Pro-(D-Lys)-OH (SEQ ID NO: 3);

[0033] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0034] In one embodiment, each of the two peptides comprises the following sequence:

[0035] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Pro)-(D-Lys)-OH (SEQ ID NO: 4);

[0036] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0037] In one embodiment, each of the two peptides comprises the following sequence:

[0038] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-NH2 (SEQ ID NO: 5),

[0039] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0040] In one embodiment, each of the two peptides comprises the following sequence:

[0041] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-OH (SEQ ID NO: 5),

[0042] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0043] In one embodiment, the peptide dimer compound or a pharmaceutically acceptable salt thereof is:

[0044]

[0045] Or its pharmaceutically acceptable salt.

[0046] In one embodiment, the peptide dimer compound or a pharmaceutically acceptable salt thereof is:

[0047]

[0048] Or its pharmaceutically acceptable salt.

[0049] In one embodiment, each of the two peptides comprises or consists of the following sequence:

[0050] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Glu)-(D-Lys)-OH (SEQ ID NO: 1),

[0051] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0052] In one embodiment, each of the two peptides comprises or consists of the following sequence:

[0053] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Gly-(D-Lys)-OH (SEQ ID NO: 2);

[0054] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0055] In one embodiment, each of the two peptides comprises or consists of the following sequence:

[0056] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Pro-(D-Lys)-OH (SEQ ID NO: 3);

[0057] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0058] In one embodiment, each of the two peptides comprises or consists of the following sequence:

[0059] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Pro)-(D-Lys)-OH (SEQ ID NO: 4);

[0060] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0061] In one embodiment, each of the two peptides comprises or consists of the following sequence:

[0062] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-NH2 (SEQ ID NO: 5),

[0063] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0064] In one embodiment, each of the two peptides comprises or consists of the following sequence:

[0065] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-OH (SEQ ID NO: 5),

[0066] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0067] Any peptide disclosed herein may contain an N-terminal Ac.

[0068] In one embodiment, the peptide dimer compound or a pharmaceutically acceptable salt thereof is:

[0069]

[0070] Or its pharmaceutically acceptable salt.

[0071] In one embodiment, the peptide dimer compound or a pharmaceutically acceptable salt thereof is:

[0072]

[0073] Or its pharmaceutically acceptable salt.

[0074] In specific embodiments of the methods disclosed herein, the peptide dimer compound or a pharmaceutically acceptable salt thereof is present in doses of approximately 100.0 mg, 112.5 mg, 125.0 mg, 137.5 mg, 150.0 mg, 162.5 mg, 175 mg, 187.5 mg, 200.0 mg, 212.5 mg, 225.0 mg, 237.5 mg, 250.0 mg, 262.5 mg, 275 mg, etc. The drug is administered to the subject at doses of 287.5 mg, 300.0 mg, 312.5 mg, 325.0 mg, 337.5 mg, 350.0 mg, 362.5 mg, 375 mg, 387.5 mg, 400.0 mg, 412.5 mg, 425.0 mg, 437.5 mg, 450.0 mg, 462.5 mg, 475 mg, 487.5 mg, or 500.0 mg. In one embodiment, the peptide dimer compound or a pharmaceutically acceptable salt thereof is administered to the subject at a dose of about 150 mg or about 450 mg. In some embodiments, the dose is administered to the subject twice daily.

[0075] In a particular embodiment, the pharmaceutically acceptable salt of the peptide dimer compound is an acetate.

[0076] In certain embodiments of the methods disclosed herein, optionally when measured at peak blood or serum levels of the antagonist, the applied dose produces an unsaturated blood receptor occupancy rate (RO%). In some embodiments, optionally when measured at peak blood or serum levels of the antagonist, the applied dose produces an RO of less than 90%, less than 80%, less than 70%, less than 60%, or less than 50%.

[0077] In a specific embodiment of the method disclosed herein, the method inhibits MadCAM1-mediated T cell proliferation in the gastrointestinal tract.

[0078] In a specific embodiment of the method disclosed herein, the method reduces the cell surface expression of β7 on CD4+ T cells in the gastrointestinal tract.

[0079] In a specific embodiment of the method disclosed herein, the method:

[0080] i) Inducing the internalization of α4β7 integrin on CD4+ T memory cells;

[0081] ii) Reduces the adhesion of MAdCAM1 to CD4+ T memory cells in the gastrointestinal tract; and / or

[0082] iii) Inhibit T cell homing to the gastrointestinal tract, optionally to the ileal lamina propia, Peyer's patch, mesenteric lymph nodes, small intestine and / or colon.

[0083] In a particular embodiment of the method disclosed herein, the IBD is ulcerative colitis.

[0084] In a particular embodiment of the method disclosed herein, the IBD is Crohn's disease.

[0085] In specific embodiments of the method disclosed herein, the method generates one or more of the following pharmacokinetic parameters in the plasma of the subject:

[0086] Cmax (ng / mL) is 1-25;

[0087] Tmax (hours) is 1-5;

[0088] AUC t (nanogram-hours / mL) is 10-250;

[0089] AUC inf (nanogram-hours / mL) is 10-300;

[0090] t 1 / 2 (Hours) is 3-10;

[0091] AUC tau (nanogram-hours / mL) is 30-130;

[0092] Ctrough (ng / mL) is 1-5;

[0093] The cumulative Cmax (ng.mL) ranged from 0.5 to 2.5; and

[0094] Cumulative AUC t (Ng·h / mL) is 0.5-3.0.

[0095] In specific embodiments of the method disclosed herein, the method generates one or more of the following pharmacokinetic parameters in the plasma of the subject:

[0096] ROmax (%) is 50-100;

[0097] receptor expression changes max (%) ranges from -20 to -60;

[0098] The mean change in receptor expression (%) ranged from -10 to -55.

[0099] The steady-state ROmax (%) is 80-100;

[0100] Average RO 0-24 (Hourly %) is 50-95;

[0101] Average RO 0-12 (Hourly %) is 80-95%; and

[0102] Average RO 12-24 (Hourly %) is 70-90%.

[0103] On the other hand, this disclosure provides a method of treating an inflammatory disease or condition in a subject of need, the method comprising administering an α4β7 integrin antagonist to the subject, wherein the antagonist is administered at a dose that optionally produces an unsaturated blood receptor occupancy (RO%) when measured at peak blood or serum levels of the antagonist. In some embodiments, the antagonist is administered at a dose that optionally produces less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% blood RO when measured at peak blood or serum levels of the antagonist. In some embodiments, the antagonist is present in a pharmaceutical composition formulated for administration via a route selected from: oral administration, parenteral administration, subcutaneous administration, oral administration, nasal administration, inhalation administration, topical administration, and rectal administration. In some embodiments, the antagonist is administered orally or rectally.

[0104] In certain embodiments of any of the disclosed methods, the inflammatory disease or condition is selected from the group consisting of: inflammatory bowel disease (IBD), adult IBD, pediatric IBD, adolescent IBD, ulcerative colitis, Crohn's disease, celiac disease (non-tropical stomatitis), enteropathy associated with seroreactive arthropathy, microscopic colitis, collagenous colitis, eosinophilic gastroenteritis, radiation therapy, chemotherapy, pouchitis following rectocele and ileoanal anastomosis, gastrointestinal cancer, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, pericholangitis, chronic bronchitis, chronic sinusitis, asthma, primary sclerosing cholangitis, HIV infection, eosinophilic asthma, eosinophilic esophagitis, gastritis, colitis, microscopic colitis, and graft-versus-host disease (GVDH). In a particular embodiment, the disease or condition is IBD, such as ulcerative colitis or Crohn's disease.

[0105] In some embodiments, the antagonist is a peptide dimer compound comprising two peptides or a pharmaceutically acceptable salt thereof.

[0106] Each of the two peptides comprises any of the following sequences (optionally including an N-terminal Ac) or consists of any of the following sequences:

[0107] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Glu)-(D-Lys)-OH (SEQ ID NO: 1);

[0108] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Gly-(D-Lys)-OH (SEQ ID NO: 2);

[0109] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Pro-(D-Lys)-OH (SEQ ID NO: 3);

[0110] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Pro)-(D-Lys)-OH (SEQ ID NO: 4);

[0111] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-OH (SEQ ID NO: 5);

[0112] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-NH2 (SEQ ID NO: 5);

[0113] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-OH (SEQ ID NO: 6);

[0114] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-NH2 (SEQ ID NO: 6);

[0115] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Pro-(D-Lys)-OH (SEQ ID NO: 7);

[0116] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Pro-(D-Lys)-NH2 (SEQ ID NO: 7);

[0117] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Pro)-(D-Lys)-OH (SEQ ID NO: 8); or

[0118] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Pro)-(D-Lys)-NH2 (SEQ ID NO: 8);

[0119] Each of the two peptides comprises: a thioether bond between a 2-methylbenzoyl group and a Pen; or a disulfide between two Pens; wherein the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and wherein the linker moiety is diethylene glycol (DIG).

[0120] In a particular embodiment, each of the two peptides comprises or is composed of the following sequences:

[0121] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Glu)-(D-Lys)-OH (SEQ ID NO: 1),

[0122] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0123] In a particular embodiment, each of the two peptides comprises or is composed of the following sequences:

[0124] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Gly-(D-Lys)-OH (SEQ ID NO: 2);

[0125] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0126] In a particular embodiment, each of the two peptides comprises or is composed of the following sequences:

[0127] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Pro-(D-Lys)-OH (SEQ ID NO: 3);

[0128] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0129] In a particular embodiment, each of the two peptides comprises or is composed of the following sequences:

[0130] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Pro)-(D-Lys)-OH (SEQ ID NO: 4);

[0131] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0132] In a particular embodiment, each of the two peptides comprises or is composed of the following sequences:

[0133] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-NH2 (SEQ ID NO: 5),

[0134] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0135] In a particular embodiment, each of the two peptides comprises or is composed of the following sequences:

[0136] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-OH (SEQ ID NO: 5),

[0137] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0138] In a particular embodiment, the peptide dimer compound or a pharmaceutically acceptable salt thereof is:

[0139]

[0140] Or its pharmaceutically acceptable salt.

[0141] In a particular embodiment, the peptide dimer compound or a pharmaceutically acceptable salt thereof is:

[0142]

[0143] Or its pharmaceutically acceptable salt.

[0144] In a particular embodiment, each of the two peptides comprises or is composed of the following sequences:

[0145] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Glu)-(D-Lys)-OH (SEQ ID NO: 1),

[0146] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0147] In a particular embodiment, each of the two peptides comprises or is composed of the following sequences:

[0148] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Gly-(D-Lys)-OH (SEQ ID NO: 2);

[0149] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0150] In a particular embodiment, each of the two peptides comprises or is composed of the following sequences:

[0151] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Pro-(D-Lys)-OH (SEQ ID NO: 3);

[0152] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0153] In a particular embodiment, each of the two peptides comprises or is composed of the following sequences:

[0154] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Pro)-(D-Lys)-OH (SEQ ID NO: 4);

[0155] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0156] In a particular embodiment, each of the two peptides comprises or is composed of the following sequences:

[0157] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-NH2 (SEQ ID NO: 5),

[0158] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0159] In a particular embodiment, each of the two peptides comprises the following sequence:

[0160] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-OH (SEQ ID NO: 5),

[0161] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0162] In a particular embodiment, the peptide dimer compound or a pharmaceutically acceptable salt thereof is:

[0163]

[0164] Or its pharmaceutically acceptable salt.

[0165] In a particular embodiment, the peptide dimer compound or a pharmaceutically acceptable salt thereof is:

[0166]

[0167] Or its pharmaceutically acceptable salt.

[0168] In certain embodiments, the peptide dimer compound or a pharmaceutically acceptable salt thereof is present in doses of approximately 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12.5 mg, 25.0 mg, 37.5 mg, 50.0 mg, 62.5 mg, 75 mg, 87.5 mg, 100.0 mg, 112.5 mg, 125.0 mg, 137.5 mg, 150.0 mg, 162.5 mg, 175 mg, 187.5 mg, 200.0 mg, 212.5 mg, and 225.0 mg. The subject was given doses of 237.5 mg, 250.0 mg, 262.5 mg, 275 mg, 287.5 mg, 300.0 mg, 312.5 mg, 325.0 mg, 337.5 mg, 350.0 mg, 362.5 mg, 375 mg, 387.5 mg, 400.0 mg, 412.5 mg, 425.0 mg, 437.5 mg, 450.0 mg, 462.5 mg, 475 mg, 487.5 mg, or 500.0 mg. In a particular embodiment, the doses were administered to the subject once or twice daily.

[0169] In a particular embodiment, the pharmaceutically acceptable salt of the peptide dimer compound is an acetate.

[0170] In a related aspect, this disclosure provides a pharmaceutical composition comprising a peptide dimer compound or a pharmaceutically acceptable salt thereof disclosed in any one of claims 39 to 58. In a particular embodiment, the composition is formulated for oral delivery, optionally wherein the composition comprises an enteric coating. In a particular embodiment, the method comprises administering the disclosed pharmaceutical composition to the subject.

[0171] In specific embodiments of the methods and compositions disclosed herein, the antagonist or a pharmaceutically acceptable salt thereof inhibits the binding of α4β7 integrin to MAdCAM1.

[0172] In specific embodiments of the methods and compositions disclosed herein, the antagonist or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, is administered to the subject in need at intervals sufficient to improve or alleviate the symptoms. In specific embodiments, the intervals are selected from the group consisting of: all day, hourly, every four hours, once daily, twice daily, three times daily, four times daily, every other day, weekly, every two weeks, and monthly. In some embodiments, the antagonist or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, is administered as an initial dose, followed by one or more subsequent doses, with a minimum interval of less than one day between any two doses, and each of said doses comprises an effective amount of the antagonist. In some embodiments, an effective amount of the antagonist or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, is sufficient to achieve at least one of the following:

[0173] a) The saturation of the MAdCAM1 binding site on the α4β7 integrin molecule is approximately 50% or greater;

[0174] b) The inhibition of α4β7 integrin expression on the cell surface is approximately 50% or greater; and

[0175] c) The saturation of the MAdCAM1 binding site on the α4β7 molecule is about 50% or greater, and the inhibition of α4β7 integrin expression on the cell surface is about 50% or greater, wherein i) the saturation is maintained for a period of time consistent with a dosing frequency of no more than twice daily; ii) the inhibition is maintained for a period of time consistent with a dosing frequency of no more than twice daily; or iii) the saturation and the inhibition are each maintained for a period of time consistent with a dosing frequency of no more than twice daily. Attached Figure Description

[0176] Figure 1 This is a table showing the proliferation of T cells in response to the indicated treatment and the inhibition of T cell proliferation by compound A or vedolizumab.

[0177] Figure 2 This demonstrates the response to CD45RO processed with anti-CD3 or anti-CD3+MAdaCAM. - Original and CD45RO + The expression of memory T cells.

[0178] Figure 3 A-sB provides evidence of increased β7 expression during consecutive proliferation cycles. Figure 3 A) and CD4 that did not split in the presence of compound A. + Reduced surface expression of β7 in T cells Figure 3 Table B).

[0179] Figure 4 This is a graph showing the reduction in surface β7 expression in five donors after treatment with compound A.

[0180] Figure 5 AC is a graph showing the release of cytokines after treatment with anti-CD3+MAdCAM1 and inhibition by compound A: IFNγ ( Figure 5 A) IL-23 Figure 5 B) and GM-CSF ( Figure 5 C).

[0181] Figure 6 AC is a graph showing the release of cytokines after treatment with anti-CD3+MAdCAM1 and inhibition by compound A: IL-10 ( Figure 6 A), IL-5 Figure 6 B) and TNFα Figure 6 C).

[0182] Figure 7 This is a graph showing the receptor occupancy (RO) % in whole blood and Pierre's nodes after administration of a specified dose of compound A. The table below the graph provides the RO%.

[0183] Figure 8 The graph above shows the RO% in whole blood and Pierre's nodes of six individual animals treated with specified amounts of compound A. The graph below shows the RO% on day 14.

[0184] Figure 9 The graphs provided show the concentrations of compound A in plasma and Pierre's nodes after administration of a specified dose of compound A (left graph) and the RO% of compound A in whole blood and Pierre's nodes after administration of a specified dose of compound A (right graph).

[0185] Figure 10 A graph is provided showing the concentrations of compound A detected in plasma and specified tissues at different time points after treatment. The graph below represents data from the graph above, plotted with an expanded scale.

[0186] Figure 11 This is a graph summarizing the various pharmacokinetic parameters of compound A after administration of a single PO dose of 30 mg / kg in mice.

[0187] Figure 12 This is a graph showing the percentage of cultured cells with specified surface markers after a given treatment. For each cell type, the four bars from left to right correspond to the treatments indicated from top to bottom on the left.

[0188] Figure 13This is a graph showing the percentage of cultured cells with specified surface markers after a given treatment. For each cell type, the four bars from left to right correspond to the treatments indicated from top to bottom on the left.

[0189] Figure 14 This is a diagram showing the expression of α4β7 cells on the surface of PBMCs treated with compound C or compound D. FMO was used as a staining control.

[0190] Figure 15 This is a diagram showing the time-dependent expression of α4β7 on the surface of CD4+ T memory cells treated with compound A at time 0.

[0191] Figure 16 This is a diagram showing the concentration-dependent expression of α4β7 on the surface of CD4+ T memory cells treated with compound A at the indicated concentration.

[0192] Figure 17 This is a diagram showing the concentration-dependent expression of α4β7 on the surface of CD4+ T memory cells treated with compound A at the indicated concentration.

[0193] Figure 18 This is a graph showing the concentration-dependent reduction in MAdCAM1 adhesion on CD4+ T memory cells treated with the indicated concentration of compound A.

[0194] Figure 19 This is a graph showing the correlation between the percentage reduction in MAdCAM1 adhesion and the percentage reduction in α4β7 expression.

[0195] Figure 20 This is a graph showing the downregulation of α4β7 expression after treatment with compound A, followed by the recovery of α4β7 expression after treatment was stopped.

[0196] Figure 21 This is a graph showing the average plasma concentration over time after a single dose of a specified amount of compound A.

[0197] Figure 22 AB shows the receptor occupancy (%) over time after a single dose of the indicated amount of compound A. Figure 22 A) and receptor expression (%) Figure 22 Figure B).

[0198] Figure 23 AB represents the mean steady-state plasma concentration of compound A after administration of 450 mg as a liquid solution or immediate-release tablet. Figure 23 A) and receptor occupancy (%) Figure 23 B) A graph showing the time progression of a graph.

[0199] Figure 24 This is a graph showing the correlation between the plasma concentration of compound A and the receptor occupancy (%) after administration of compound A.

[0200] Figure 25 This is a graph showing the average receptor occupancy (%) in whole blood and Pierre nodes after administration of a specified dose of compound A. Corresponding values ​​are provided in the table.

[0201] Figure 26 A graph showing the dose-dependent concentrations of compound A in plasma (left) and Pierre's nodes (right) after administration of a specified dose of compound A is provided.

[0202] Figure 27 This is a table showing the receptor occupancy in individual animals after treatment with a specified dose of compound A. Detailed Implementation

[0203] Ulcerative colitis is a chronic inflammatory bowel disease (IBD) characterized by remission and relapse, and is marked by bloody diarrhea, abdominal cramps, and fatigue. Its pathogenesis is believed to be caused by an inappropriate immune response to gastrointestinal antigens and environmental triggers in genetically susceptible individuals. The highest prevalence has been reported in Europe and North America. Ulcerative colitis has a significant negative impact on patients' quality of life and places a high economic burden on healthcare systems.

[0204] Inflammatory bowel diseases, such as ulcerative colitis, have been managed with corticosteroids, 5-aminosalicylates, and immunosuppressants, and more recently, biologics targeting specific inflammatory mediators have been used. Long-term treatment options for ulcerative colitis are limited. 5-aminosalicylates, such as sulfasalazine, olsalazine, balsalazide, and various forms of mesalamine (e.g., Asacol, Pentasa, Lialda, Canasa) are only effective for mild to moderate disease, while biologics can be initiated in patients with severe disease. Several monoclonal antibodies against TNF-α (e.g., infliximab, adalimumab, golimumab, and certolizumab) are now available. Drugs targeting other cytokines involved in the inflammatory response, such as ustekinumab for IL-12 / IL-23 and the pan-JAK inhibitor tofacitinib, are now part of the treatment options available for inflammatory bowel disease, while several IL-23 and S1P1 inhibitors are currently in clinical trials.

[0205] Despite a wide range of treatment options, treatment for inflammatory bowel disease remains limited, and the available agents are not without risks. TNF-α inhibitors are ineffective in approximately one-fifth to one-third of patients, and 10%–15% of treated patients who initially showed benefit may lose response each year. Skin reactions are also the most common adverse reaction to anti-TNF therapy. These include injection site reactions, skin infections, immune-mediated complications such as psoriasis and lupus-like syndromes, and, rarely, skin cancer. Tofacitinib may increase the risk of infection and may increase the risk of thrombotic or thromboembolic events. There is a growing recognition that reducing the local inflammatory response may be promising. Oral administration of budesonide and 5-ASA is locally effective, and various other locally acting agents, including AMT-101 (an orally administered biofusion protein with local action of interleukin-10) and TD-1473 (a JAK inhibitor), have shown promise or are undergoing clinical trials. Local delivery via oral administration can allow for the delivery of higher doses of the drug to the target site without increasing systemic side effects.

[0206] Integrins are heterodimers that function as cell adhesion molecules. α4-integrins α4β1 and α4β7 are known to play important roles in lymphocyte migration throughout the gastrointestinal tract. These integrins are expressed on most leukocytes, including B and T lymphocytes, monocytes, and dendritic cells, and mediate cell adhesion at these leukocytes by binding to their respective major ligands, namely vascular cell adhesion molecule (VCAM) and mucosal addressing cell adhesion molecule 1 (MAdCAM1). VCAM and MAdCAM1 have different binding specificities because VCAM binds to both α4β1 and α4β7, while MAdCAM1 is highly specific for α4β7.

[0207] α4β7 integrin, which is primarily involved in recruiting leukocytes to the gastrointestinal (GI) tract, is present on the cell surface of a small subset of circulating T and B lymphocytes. Its major ligand, MAdCAM1, is selectively expressed on the endothelium of the intestinal vascular system and is present at increased concentrations in inflamed tissues.

[0208] This disclosure provides a method for treating IBD by inhibiting α4β7 integrin, for example, using a peptide dimer antagonist of α4β7 integrin, including, but not limited to, any peptide dimer antagonists disclosed herein. Specifically, this disclosure provides an oral dose of an α4β7 integrin antagonist effective for treating IBD, comprising ulcerative colitis. Additionally, this disclosure provides pharmacokinetic and pharmacodynamic parameters of the α4β7 integrin antagonist associated with the biological activity of the antagonist, such as inhibition of MAdCAM1-mediated T cell proliferation, reduced T cell expression of β7 (and α4β7 integrin), internalization of α4β7 integrin on T cells, reduced T cell homing to gastrointestinal tissues, reduced cytokine release by T cells, reduced T cell adhesion to MAdCAM1, and reduced gastrointestinal inflammation. In a particular embodiment, the T cells are CD4+ T memory cells.

[0209] Furthermore, it has been previously believed that the underlying mechanism for treating IBD with α4β7 integrin antagonists involves the binding of the antagonist to α4β7 expressed on circulating T cells. This prevents T cells from binding to MAdCAM1 expressed on GI endothelial cells, thereby preventing T cell extravascular migration into the inflamed gastrointestinal mucosa of IBD patients. Therefore, the goal is to achieve maximum blood receptor occupancy (RO%), for example, greater than 80%, greater than 90%, or close to 100% RO, to prevent T cells from binding to and migrating into the inflamed gastrointestinal mucosa.

[0210] In contrast, the inventors have identified an alternative mechanism by which α4β7 integrin antagonists exert local effects to inhibit inflammation in inflamed tissues, such as the inflamed gastrointestinal mucosa. As disclosed in the appended examples, when present in inflamed tissue, α4β7 integrin antagonists can inhibit MAdCAM1-mediated CD4+ T cell proliferation and cytokine production through direct binding to and stimulation of α4β7 integrin. This document demonstrates that such local effects do not require blood receptor saturation, but rather that oral administration of a subsaturated dose of the antagonist is sufficient to achieve therapeutic effects, such as endoscopic or histological improvements. Therefore, this disclosure particularly provides a method for treating IBD comprising orally administering an α4β7 integrin antagonist to a subject, comprising, but not limited to, a subsaturated blood receptor occupancy of the peptide dimer compounds disclosed herein.

[0211] In some respects, this disclosure provides methods for using α4β7 antagonist thioether peptide monomers and dimers as anti-inflammatory agents and / or immunosuppressants, for example, for treating diseases associated with the biological function of α4β7 or cells or tissues expressing MAdCAM1.

[0212] Various aspects of this invention relate to cyclized disulfide or thioether peptide compounds exhibiting integrin antagonist activity, i.e., exhibiting high specificity for α4β7 integrin. In some embodiments, each peptide of this invention comprises a downstream natural or non-natural amino acid capable of forming a cyclized structure via a disulfide or thioether bond and an upstream modified amino acid or aromatic group. The peptides of this invention exhibit increased stability when administered orally as a therapeutic agent.

[0213] In other related embodiments, the present invention provides a method for treating or preventing a disease or symptom associated with the biological function of integrin α4β7, the method comprising providing an effective amount of the peptide molecule of the present invention or the pharmaceutical composition of the present invention to a subject in need. In some embodiments, the disease or symptom is inflammatory bowel disease. In particular embodiments, the inflammatory bowel disease is ulcerative colitis or Crohn's disease. In particular embodiments, the peptide molecule inhibits the binding of α4β7 to MAdCAM1. In some embodiments, the peptide molecule or the pharmaceutical composition is provided to the subject in need at intervals sufficient to alleviate the symptom. In some embodiments, the interval is selected from the group consisting of: all day, hourly, every four hours, once a day, twice a day, three times a day, four times a day, every other day, weekly, every two weeks, and monthly. In particular embodiments, the peptide molecule or pharmaceutical composition is provided as an initial dose, followed by one or more subsequent doses, and the minimum interval between any two doses is a period of less than one day, and each of said doses comprises an effective amount of the peptide molecule. In certain embodiments, the effective amount of the peptide molecule or the pharmaceutical composition is sufficient to achieve at least one of the following: a) saturation of the MAdCAM1 binding site on the α4β7 integrin molecule at about 50% or greater; b) inhibition of α4β7 integrin expression on the cell surface at about 50% or greater; and c) saturation of the MAdCAM1 binding site on the α4β7 molecule at about 50% or greater, and inhibition of α4β7 integrin expression on the cell surface at about 50% or greater, wherein i) the saturation is maintained for a period of time consistent with a dosing frequency of no more than twice daily; ii) the inhibition is maintained for a period of time consistent with a dosing frequency of no more than twice daily; or iii) the saturation and the inhibition are each maintained for a period of time consistent with a dosing frequency of no more than twice daily. In some embodiments, the peptide molecule is administered orally, parenterally, or topically.

[0214] definition

[0215] As used herein, unless the context clearly indicates otherwise, the singular forms “a / a kind,” “and,” and “the” contain plural indicators.

[0216] When the term "comprising" is used herein, it should be understood that the invention also includes the same embodiments, wherein the term "comprising" is replaced by "consistently consisting of" or "consisting of".

[0217] As used in this specification, the following terms have the meanings indicated therein:

[0218] As used herein, the term "peptide" broadly refers to a structure comprising a sequence of two or more amino acids linked together by peptide bonds. In specific embodiments, "peptide" refers to a sequence of two or more amino acids linked together by peptide bonds. It should be understood that this term does not indicate a specific length of the polymer of amino acids, nor is it intended to imply or distinguish whether a polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. As generally used herein, the term "peptide" includes both peptide monomers and peptide dimers.

[0219] As used herein, the term "monomer" may also be referred to as "peptide monomer," "peptide monomer molecule," or "monomer peptide." The term "monomer" refers to a single sequence of two or more amino acids linked together by peptide bonds.

[0220] As used herein, the term "dimer" broadly refers to a peptide comprising two monomeric peptide subunits (e.g., thioether monomeric peptides) linked at respective C-termini or N-termini. The dimers of the present invention may comprise homodimers or heterodimers that act as integrin antagonists. The term "dimer" may also be referred to herein as "peptide dimer," "peptide dimer molecule," "dimeric peptide," or "dimeric compound." The term "monomeric peptide subunit" may also be referred herein as "monomer subunit," "peptide monomer subunit," "peptide subunit," "peptide dimer subunit," "dimer subunit," "monomer subunit," or "subunit of peptide dimer."

[0221] As used herein, the term "thioether" refers to a cyclic covalent bond, or CS bond, formed between an upstream amino acid or aromatic acid group and a downstream sulfur-containing amino acid or its isostere.

[0222] As used herein, the term “linker” broadly refers to a chemical structure that can link two thioether monomer subunits together to form a dimer.

[0223] As used herein, the term "L-amino acid" refers to the "L" isomer of a peptide, and conversely, the term "D-amino acid" refers to the "D" isomer of a peptide. The amino acid residues described herein are preferably in the "L" isomer form; however, residues in the "D" isomer form may substitute for any L-amino acid residue, provided the peptide retains its desired function.

[0224] Unless otherwise indicated, as used herein, the term "NH2" refers to a free amino group present at the amino terminus of a polypeptide. As used herein, the term "OH" refers to a free carboxyl group present at the carboxyl terminus of a peptide. Further, as used herein, the term "Ac" refers to acetyl protection via acylation at the N-terminus of a polypeptide. Where indicated, "NH2" refers to the free amino side chain of an amino acid. Where indicated, as used herein, the term "Ac" refers to acylation of an amino acid with an NH2 group.

[0225] As used in this article, the term "carboxyl group" refers to -CO2H.

[0226] As used herein, the term "isostere" or "isostere substitution" refers to any amino acid or other similar moiety having chemical and / or structural properties similar to a particular amino acid. In specific embodiments, an "isostere" or "suitable isostere" of an amino acid is another amino acid in the same class, wherein the amino acid belongs to the following classes based on the tendency of its side chain to contact polar solvents such as water: hydrophobic (low tendency to contact water), polar, or charged (energy-favorable contact with water). Charged amino acid residues include lysine (+), arginine (+), aspartic acid (-), and glutamic acid (-). Polar amino acids include serine, threonine, asparagine, glutamine, histidine, and tyrosine. Hydrophobic amino acids include alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, cysteine, and methionine. The amino acid glycine has no side chain and is difficult to classify into one of the above classes. However, glycine is typically found on the protein surface, usually within a loop, thus providing high flexibility to these regions, and isosteres can possess similar characteristics. Proline has the opposite effect, thereby providing rigidity to the protein structure by imposing a certain twist angle on fragments of the polypeptide chain.

[0227] As used herein, the term "cyclization" refers to a reaction in which a portion of a polypeptide molecule is linked to another portion of the polypeptide molecule, such as by forming a disulfide bond or thioether bond, to form a closed ring. In certain embodiments, the monomeric subunits of the peptide monomers and peptide dimers of the present invention are cyclized via intramolecular disulfide bonds or thioether bonds.

[0228] As used herein, the term "receptor" refers to a molecular chemical group on or inside a cell surface that has an affinity for a specific chemical group or molecule. The binding between peptide molecules and targeting integrins can provide useful diagnostic tools.

[0229] As used herein, the term "integrin-related disease" refers to an indication that results from integrin binding and that can be treated with the administration of an integrin antagonist.

[0230] As used herein, the term "pharmaceutically acceptable salt" refers to a salt or zwitterionic form of a compound of the present invention, wherein the pharmaceutically acceptable salt is water-soluble, oil-soluble, or dispersible, suitable for treating diseases without adverse toxicity, irritation, or allergic responses; commensurate with a reasonable benefit / risk ratio, and effective for its intended use. The salt may be prepared during the final isolation and purification of the compound or separately by reacting the amino group with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, diglucose, glyceryl phosphate, hemisulfate, heptanate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (hydroxyethyl sulfonate), lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthalenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, picrate, neopentanoate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate. Similarly, the amino groups in the compounds of the present invention can be quaternized using methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and dipentyl sulfate; decyl, lauryl, myristyl, and sterol chlorides, bromides, and iodides; and benzyl bromide and phenethyl bromide. Examples of acids that can be used to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid.

[0231] As used herein, the term “N(α)methylation” describes the methylation of an α-amine of an amino acid, and is also commonly referred to as N-methylation.

[0232] As used herein, the term "acylated organic compound" refers to a variety of compounds having carboxylic acid functionality that can be used to acylate the C-terminus and / or N-terminus of a peptide molecule. Non-limiting examples of acylated organic compounds include cyclopropylacetic acid, 4-fluorobenzoic acid, 4-fluorophenylacetic acid, 3-phenylpropionic acid, succinic acid, glutaric acid, cyclopentanecarboxylic acid, glutaric acid, succinic acid, 3,3,3-trifluoropropionic acid, and 3-fluoromethylbutyric acid.

[0233] All peptide sequences are written according to generally accepted convention, with the α-N-terminal amino acid residue on the left and the α-C-terminus on the right. As used herein, the term "α-N-terminus" refers to the free α-amino group of an amino acid in the peptide, and the term "α-C-terminus" refers to the free α-carboxylic acid terminus of an amino acid in the peptide.

[0234] As used herein, the term "amino acid" or "any amino acid" refers to any and all amino acids, including naturally occurring amino acids (e.g., α-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. The term "amino acid" includes both D-amino acids and L-amino acids. Natural amino acids include those found in nature, such as the 23 amino acids that combine into peptide chains to form the building blocks of a wide range of proteins. These natural amino acids are primarily L stereoisomers, although some D-amino acids are found in bacterial envelopes and some antibiotics. "Non-standard" natural amino acids are pyrrolidone (found in methanogens and other eukaryotes), selenocysteine ​​(found in many non-eukaryotes and most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). "Unnatural / non-natural" amino acids are non-proteinogenic amino acids that are either naturally occurring or chemically synthesized (i.e., those not naturally encoded or not found in the genetic code). More than 140 naturally occurring amino acids are known, and thousands more combinations are possible. Examples of “non-natural” amino acids include β-amino acids (β-amino acids). 3 and β 2 This includes homologous amino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, cyclically substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, α-methyl amino acids, and N-methyl amino acids. Non-natural amino acids also include modified amino acids. "Modified" amino acids include those that have been chemically modified to include one or more groups or chemical moieties that are not naturally present on the amino acid (e.g., natural amino acids).

[0235] Generally, the names of naturally occurring and non-natural aminoacyl residues used herein follow the naming conventions recommended by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature, such as those set forth in "Nomenclature of α-Amino Acids (Recommendations), 1974" in *Biochemistry*, 14(2), (1975). The extent to which the names and abbreviations of amino acids and aminoacyl residues used in this specification and the appended claims differ from these recommendations will be clearly explained to the reader. Some abbreviations used to describe the invention are defined in Table 1 below.

[0236] Table 1. Abbreviations

[0237]

[0238]

[0239]

[0240]

[0241]

[0242] peptide antagonists

[0243] This invention generally relates to cyclic peptides, such as disulfides and thioether peptides, that have been shown to have integrin antagonist activity. Specifically, this invention relates to various peptides that form cyclic structures via intramolecular bonds, such as disulfide bonds or thioether bonds. Although this disclosure generally relates to peptides having disulfide intramolecular bonds or thioether intramolecular bonds, it should be understood that other cyclic peptide antagonists of α4b7 integrin, including those comprising intramolecular bonds of different properties, and cyclic peptide antagonists of α4β7 integrin (including bonds between two peptide monomer subunits) can also be used to practice the methods disclosed herein. Some embodiments relate to disulfide or thioether peptide monomers having integrin antagonist activity. Some embodiments relate to disulfide or thioether peptide dimers having integrin antagonist activity, comprising heteromonomers or homomonomers of thioether peptide subunits, wherein the disulfide or thioether peptide subunits are linked at their C-terminus or N-terminus. As discussed below, cyclization of peptides, peptide monomers, or peptide subunits has been shown to increase the potency, selectivity, and stability of peptide molecules. In some embodiments, dimerization of peptide monomers increases efficiency, selectivity, and / or stability compared to non-dimerized peptides. Illustrative peptides and their genera are provided in the following patent application publications, each of which is incorporated herein by reference in its entirety: PCT Application Publications WO 2014 / 059213, WO 2014 / 165448, WO 2014 / 165449, WO 2015 / 176035, WO 2016 / 054411, and WO 2016 / 054445.

[0244] In some instances, the monomeric peptide further includes a C-terminus and / or an N-terminus comprising a free amine (or both a C-terminus and an N-terminus comprising a free amine). Similarly, the peptide dimer may include one or more C-termini or N-termini comprising a free amine. Thus, a user can modify either end to include a modifying group, such as PEGylation, for example, small PEGylation (e.g., PEG4-PEG13). A user can further modify either end by acylation. For example, in some instances, at least one of the N-terminus and C-terminus of the peptide molecule is acylated with an acylated organic compound selected from the group consisting of: 2-Me-trifluorobutyl, trifluoropentyl, acetyl, octyl, butyl, pentyl, hexyl, palmityl, trifluoromethylbutyric acid, cyclopentanecarboxylic acid, cyclopropylacetic acid, 4-fluorobenzoic acid, 4-fluorophenylacetic acid, and 3-phenylpropionic acid. In some instances, the peptide molecule of the present invention includes both a free carboxyl terminus and a free amino terminus, thereby allowing a user to selectively modify the peptide to achieve the desired modification. It should be further understood that, unless otherwise indicated, the C-terminal residues of the thioether peptides (e.g., thioether monomers) disclosed herein are amides or acids. Therefore, those skilled in the art will understand that the thioether peptides of the present invention can be selectively modified as needed.

[0245] Regarding peptide dimers, it should be understood that monomeric subunits dimerize to form peptide dimer molecules, for example, by linking or dimerizing monomeric subunits through suitable linker moieties as defined herein. Some monomeric subunits exhibit having both a C-terminus and an N-terminus comprising a free amine. Therefore, a user can modify either end of a monomeric subunit to eliminate the C-terminal or N-terminal free amine, thereby allowing dimerization at the remaining free amine. Thus, some monomeric subunits include both a free carboxyl or amide at the C-terminus and a free amino terminus, thereby allowing a user to selectively modify the subunit to achieve dimerization at the desired terminus. Therefore, those skilled in the art will understand that the monomeric subunits of the present invention can be selectively modified to achieve a single, specific amine for the desired dimerization.

[0246] It should be further understood that, unless otherwise indicated, the C-terminal residues of the monomer subunits disclosed herein include -OH or -NH2. Furthermore, it should be understood that, as is generally understood in the art, dimerization at the C-terminus can be facilitated by using suitable amino acids in which the side chains have amine functionality. In particular embodiments, the linker binds to the functional amine group in the C-terminal amino acid of each peptide monomer subunit to form a dimer. Regarding the N-terminal residues, it should generally be understood that dimerization can be achieved by the free amine of the terminal residue, or by using suitable amino acid side chains having a free amine, as is generally understood in the art.

[0247] The peptide monomers and dimers of the present invention, or their peptide subunits, may further include one or more terminal modifying groups. In at least one embodiment, the ends of the peptide are modified to include terminal modifying groups selected from the non-limiting group consisting of: DIG, PEG4, PEG13, PEG25, PEG1K, PEG2K, PEG4K, PEG5K, polyethylene glycol with a molecular weight of 400 Da to 40,000 Da, PEG with a molecular weight of 40,000 Da to 80,000 Da, IDA, ADA, glutaric acid, succinic acid, isophthalic acid, 1,3-phenylene diacetic acid, 1,4-phenylene diacetic acid, 1,2-phenylene diacetic acid, AADA, and suitable aliphatic, aromatic, and heteroaromatic compounds.

[0248] In some embodiments of the peptide dimer, peptide dimer subunit, or peptide monomer described herein, the N-terminus further includes a suitable linker moiety or other modifying group. In some embodiments of the peptide monomer described herein, the N-terminus may be further acylated.

[0249] Table 2 provides non-limiting examples of terminal modification groups.

[0250] Table 2. Illustrative terminal modification groups

[0251]

[0252]

[0253] The linker portion of the present invention may comprise any structure, length, and / or size compatible with the teachings herein. In at least one embodiment, the linker portion is selected from the non-limiting group consisting of: DIG, PEG4, PEG4-Biotin, PEG13, PEG25, PEG1K, PEG2K, PEG3.4K, PEG4K, PEG5K, IDA, ADA, Boc-IDA, glutaric acid, isophthalic acid, 1,3-phenylene diacetic acid, 1,4-phenylene diacetic acid, 1,2-phenylene diacetic acid, triazine, Boc-triazine, IDA-Biotin, PEG4-Biotin, AADA, suitable aliphatic compounds, aromatic compounds, heteroaromatic compounds, and polyethylene glycol-based linkers with a molecular weight of about 400 Da to about 40,000 Da or about 40,000 Da to about 80,000 Da.

[0254] When the linker is IDA, ADA, or any linker having a free amine, the linker can be acylated with an acylated organic compound selected from the group consisting of: 2-me-trifluorobutyl, trifluoropentyl, acetyl, octyl, butyl, pentyl, hexyl, palmityl, lauryl, oleoyl, lauryl, trifluoromethylbutyric acid, cyclopentanecarboxylic acid, cyclopropylacetic acid, 4-fluorobenzoic acid, 4-fluorophenylacetic acid, 3-phenylpropionic acid, tetrahydro-2H-pyran-4-carboxylic acid, succinic acid and glutaric acid, straight-chain fatty acids having 10 to 20 carbon units, cholic acids, and other bile acids. In some instances, small PEGs (PEG4-PEG13), Glu, or Asp are used as spacers before acylation.

[0255] In some embodiments, linkers connect two monomer subunits by linking two sulfur-containing C-terminal or N-terminal amino acids. In some embodiments, the two sulfur-containing amino acids are linked by linkers comprising dihalides, aliphatic chains, or PEG. In some embodiments, linkers connect two monomer subunits by linking a sulfur-containing C-terminal amino acid at the C-terminus of each monomer subunit. In some embodiments, the two sulfur-containing amino acids are linked by linkers comprising: a bifunctional maleimide crosslinker, a dihalide, 1,2-bis(bromomethyl)benzene, 1,2-bis(chloromethyl)benzene, 1,3-bis(bromomethyl)benzene, 1,3-bis(chloromethyl)benzene, 1,4-bis(bromomethyl)benzene, 1,4-bis(chloromethyl)benzene, 3,3'-bis-bromomethyl-biphenyl, or 2,2'-bis-bromomethyl-biphenyl. Specific haloacetyl crosslinkers contain iodoacetyl or bromoacetyl groups. These bifunctional linkers may contain spacers comprising PEG or aliphatic chains.

[0256] Table 3 provides unrestricted examples of suitable connector sub-parts.

[0257] Table 3. Explanatory Connector Subsections

[0258]

[0259]

[0260]

[0261]

[0262] Those skilled in the art will understand that certain amino acids and other chemical moieties are modified when bound to another molecule. For example, an amino acid side chain can be modified when it forms an intramolecular bridge with another amino acid side chain. Additionally, the Cl moiety is released when Homo-Ser-Cl binds to an amino acid such as Cys or Pen via a thioether bond. Therefore, as used herein, references are made to the peptide dimers present in the present invention (e.g., at position Xaa). 4 Or location Xaa 10 Amino acids or modified amino acids, such as Homo-Ser-Cl, are intended to be included in the peptide in the form of such amino acids or modified amino acids present both before and after the formation of intramolecular bonds.

[0263] In certain embodiments, although it should be understood that the methods disclosed herein may be practiced with other peptide antagonists, including those disclosed in the PCT applications incorporated herein by reference, the methods disclosed herein may be practiced with any of the following peptide antagonists of α4β7 integrin.

[0264] In some embodiments, the peptide antagonist is a peptide dimer compound comprising two peptides or a pharmaceutically acceptable salt thereof; wherein each of the two peptides comprises or is composed of any of the following sequences:

[0265] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Glu)-(D-Lys)-OH (SEQ ID NO: 1);

[0266] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Gly-(D-Lys)-OH (SEQ ID NO: 2);

[0267] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Pro-(D-Lys)-OH (SEQ ID NO: 3);

[0268] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Pro)-(D-Lys)-OH (SEQ ID NO: 4);

[0269] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-OH (SEQ ID NO:5); or

[0270] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-NH2 (SEQ ID NO:5);

[0271] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-OH (SEQ ID NO:6);

[0272] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-NH2 (SEQ ID NO:6);

[0273] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Pro-(D-Lys)-OH (SEQ ID NO:7);

[0274] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Pro-(D-Lys)-NH2 (SEQ ID NO:7);

[0275] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Pro)-(D-Lys)-OH (SEQ ID NO:8); or

[0276] Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Pro)-(D-Lys)-NH2 (SEQ ID NO:8);

[0277] Each of the two peptides comprises: a thioether bond between a 2-methylbenzoyl group and a Pen; or a disulfide between two Pens; wherein the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and wherein the linker moiety is diethylene glycol (DIG). The peptide may also contain an N-terminal Ac.

[0278] In any specific embodiment of the peptide antagonist or its pharmaceutically acceptable salt, the pharmaceutically acceptable salt of the peptide dimer compound is an acetate.

[0279] In some embodiments, each of the two peptides comprises the following sequence:

[0280] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Glu)-(D-Lys)-OH (SEQ ID NO: 1),

[0281] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0282] In some embodiments, each of the two peptides comprises the following sequence:

[0283] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Gly-(D-Lys)-OH (SEQ ID NO: 2);

[0284] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0285] In some embodiments, each of the two peptides comprises the following sequence:

[0286] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Pro-(D-Lys)-OH (SEQ ID NO: 3);

[0287] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0288] In some embodiments, each of the two peptides comprises or consists of the following sequences:

[0289] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Pro)-(D-Lys)-OH (SEQ ID NO: 4);

[0290] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0291] In some embodiments, each of the two peptides comprises or consists of the following sequences:

[0292] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-NH2 (SEQ ID NO: 5),

[0293] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0294] In some embodiments, each of the two peptides comprises or consists of the following sequences:

[0295] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-OH (SEQ ID NO: 5),

[0296] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0297] In some embodiments, the peptide dimer compound or a pharmaceutically acceptable salt thereof is:

[0298]

[0299] Or its pharmaceutically acceptable salt.

[0300] In some embodiments, the peptide dimer compound or a pharmaceutically acceptable salt thereof is:

[0301]

[0302] Or its pharmaceutically acceptable salt.

[0303] In some embodiments, each of the two peptides comprises or consists of the following sequences:

[0304] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Glu)-(D-Lys)-OH (SEQ ID NO: 1),

[0305] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0306] In some embodiments, each of the two peptides comprises or consists of the following sequences:

[0307] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Gly-(D-Lys)-OH (SEQ ID NO: 2);

[0308] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0309] In some embodiments, each of the two peptides comprises or consists of the following sequences:

[0310] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-Pro-(D-Lys)-OH (SEQ ID NO: 3);

[0311] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0312] In some embodiments, each of the two peptides comprises or consists of the following sequences:

[0313] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Pro)-(D-Lys)-OH (SEQ ID NO: 4);

[0314] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0315] In some embodiments, each of the two peptides comprises or consists of the following sequences:

[0316] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-NH2 (SEQ ID NO: 5);

[0317] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0318] In some embodiments, each of the two peptides comprises or consists of the following sequences:

[0319] 2-Methylbenzoyl-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-OH (SEQ ID NO: 5),

[0320] Each of the two peptides comprises a thioether bond between a 2-methylbenzoyl group and a pen, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, and the linker moiety is diethylene glycol (DIG).

[0321] In some embodiments, the peptide dimer compound or a pharmaceutically acceptable salt thereof is:

[0322]

[0323] Or its pharmaceutically acceptable salt.

[0324] In some embodiments, the peptide dimer compound or a pharmaceutically acceptable salt thereof is:

[0325]

[0326] Or its pharmaceutically acceptable salt.

[0327] In certain embodiments, as described in the appended examples, the peptide dimer compound is compound A or compound B.

[0328] Peptide bioactivity

[0329] In some embodiments, the peptide molecules disclosed herein exhibit increased affinity for α4β7, increased selectivity for α4β1, and increased stability in simulated intestinal fluid (SIF) and in the gastric environment under reducing conditions. These novel antagonist molecules demonstrate a high binding affinity for α4β7, thereby preventing the binding between α4β7 and the MAdCAM1 ligand. Therefore, in various experiments, these peptide molecules have been shown to effectively eliminate and / or reduce inflammatory processes.

[0330] Peptide monomers and dimer molecules bind to or associate with α4β7 integrin to disrupt or block the binding between α4β7 and the MAdCAM1 ligand. In some embodiments, the peptide dimers and monomer molecules of the present invention inhibit or reduce the binding between α4β7 and the MAdCAM1 ligand. In some embodiments, the peptides of the present invention reduce the binding between α4β7 and the MAdCAM1 ligand by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a negative control peptide. Methods for determining the binding are known in the art and described herein, and include, for example, ELISA assays.

[0331] In some embodiments, the IC50 of the peptide monomer or dimer molecule is <500 nM, <250 nM, <100 nM, <50 nM, <25 nM, or <10 nM. The method for determining activity is any method known in the art and includes those described in the appended examples.

[0332] In some embodiments, the peptide monomer or dimer molecules have a half-life greater than 180 minutes when exposed to simulated intestinal fluid (SIF). Some embodiments further provide peptide monomer or dimer molecules with a half-life ranging from approximately 1 minute to approximately 180 minutes. Similarly, when tested in a DTT (dithiothreitol) assay, these peptides are stable to the gastric environment under reduced conditions and with a half-life >120 minutes.

[0333] In some embodiments, the peptide monomer or dimer molecule exhibits increased stability, increased gastrointestinal stability, and / or increased stability in irritated intestinal fluid (SIF) compared to a control peptide. In specific embodiments, the control peptide is a peptide having the same or highly correlated amino acid sequence as the peptide monomer or dimer molecule (e.g., >90% sequence identity) but not forming a cyclized structure via a thioether bond. In some embodiments involving a dimer molecule, the control peptide is not dimerized. In specific embodiments, the only difference between the peptide monomer or dimer molecule and the control peptide is that the peptide includes one or more amino acid substitutions that introduce one or more amino acid residues into the peptide, wherein the introduced residues form a thioether bond with another residue in the peptide.

[0334] Methods for determining the stability of peptides are known in the art. In some embodiments, a SIF assay is used to determine the stability of peptides (e.g., peptide monomers or dimers described herein), for example, as described in the appended examples. In specific embodiments, the peptide monomers or dimers of the present invention, when exposed to SIF, have a half-life greater than 1 minute, greater than 10 minutes, greater than 20 minutes, greater than 30 minutes, greater than 60 minutes, greater than 90 minutes, greater than 120 minutes, greater than 3 hours, or greater than 4 hours under a given set of conditions (e.g., temperature). In some embodiments, the temperature is about 25°C, about 4°C, or about 37°C, and the pH is physiological pH or about 7.4.

[0335] In some embodiments, the half-life is measured in vitro using any suitable method known in the art. For example, in some embodiments, the stability of the peptide monomer or dimer molecules of the present invention is determined by incubating the peptide with pre-warmed human serum (Sigma) at 37°C. Samples are taken at different time points, typically up to 24 hours, and the stability of the samples is analyzed by isolating the peptide monomer or dimer from serum proteins and then analyzing the presence of the peptide monomer or dimer of interest using LC-MS.

[0336] In some embodiments, the peptide dimer or monomer molecule inhibits or reduces α4β7-mediated inflammation. In related embodiments, the peptide monomer or dimer of the present invention inhibits or reduces the secretion or release of one or more α4β7-mediated cytokines (including any cytokines disclosed herein) by T cells, such as GI mucosa, in response to MAdCAM1. Methods for measuring inhibition of cytokine secretion and inhibition of signaling molecules are known in the art.

[0337] In some embodiments, the peptide monomer or dimer molecule exhibits increased binding selectivity. In some embodiments, the affinity of the peptide monomer or dimer for α4β7 is at least two, three, five, or ten times that for α4β1.

[0338] In some embodiments, peptide monomers or dimer molecules exhibit increased potency due to the substitution of various natural aminoacyl residues with N-methylated analog residues. In certain embodiments, potency is measured as the IC50 of binding to α4β7, for example, as determined as described herein, while in some embodiments, potency indicates functional activity, for example, based on cell adhesion assays.

[0339] In certain embodiments, any of these superior properties of the peptides of the present invention compared to control peptides were determined.

[0340] Manufacturing method

[0341] The peptides (e.g., peptide monomers or peptide dimers) of the present invention can be synthesized using techniques known to those skilled in the art, such as those disclosed in PCT applications WO 2014 / 059213, WO 2014 / 165448, WO 2014 / 165449, WO 2015 / 176035, WO 2016 / 054411, or WO 2016 / 054445. Such techniques involve using commercially available robotic protein synthesizers (e.g., the Symphony multipeptide synthesizer from Protein Technologies). In some embodiments, novel peptide monomers or dimer subunits are synthesized and purified using the techniques described herein.

[0342] Treatment methods and pharmaceutical compositions

[0343] In some embodiments, the present invention provides a method for treating an individual or subject suffering from a condition or indication characterized by the binding of α4β7 integrin, for example, to MAdCAM1, wherein the method comprises providing or administering to the individual or subject an integrin antagonist, such as a peptide molecule, as described herein. In particular embodiments, the subject or individual is a mammal, such as a human, or a non-human mammal, such as a dog, cat, or horse. It should be understood that the integrin antagonist may be present in pharmaceutical compositions, such as any of the compositions disclosed herein. It should be further understood that other agents that inhibit or disrupt α4β7 integrin or MAdCAM1 signaling or the binding of α4β7 integrin to, for example, MAdCAM1 may be used as alternatives to the antagonists disclosed herein.

[0344] In some embodiments of the disclosed method, the method reduces the cell surface expression of β7 on CD4+ T cells in the gastrointestinal tract.

[0345] In some embodiments of the disclosed method, the method inhibits MadCAM1-mediated T cell proliferation in the gastrointestinal tract.

[0346] In some embodiments of the disclosed method, the method reduces the cell surface expression of β7 on CD4+ T cells in the gastrointestinal tract.

[0347] In some embodiments of the disclosed method, the method induces the internalization of α4β7 integrin on CD4+ T memory cells.

[0348] In some embodiments of the disclosed method, the method reduces the adhesion of MAdCAM1 to CD4+ T memory cells in the gastrointestinal tract.

[0349] In some embodiments of the disclosed method, the method inhibits T cell homing to the gastrointestinal tract, optionally to the lamina propria of the ileum and / or Pierre's nodes.

[0350] In some embodiments of the disclosed method, the method is used to treat IBD, optionally wherein the IBD is ulcerative colitis or Crohn's disease.

[0351] In some embodiments of the disclosed method, the method generates one or more of the following pharmacokinetic parameters in the plasma of the subject:

[0352] Cmax (ng / mL) is 1-25, optionally 4-12;

[0353] Tmax (hours) is 1-5, and can be selected as 2-4;

[0354] AUC t(nanogram-hours / mL) is 10-250, optionally 50-150;

[0355] AUC inf (nanogram-hours / mL) is 10-300, optionally 30-250;

[0356] t 1 / 2 (Hours) is 3-10, and optional locations are 4-10;

[0357] AUC tau (nanogram-hours / mL) is 30-130;

[0358] Ctrough (ng / mL) is 1-5;

[0359] The cumulative Cmax (ng.mL) was 0.5–2.5, optionally 2–3; and

[0360] Cumulative AUC t (Ng·h / mL) is 0.5-3.0.

[0361] In specific embodiments of these methods, the methods include orally administering the antagonist disclosed herein, optionally compound A, at a dose of about 150 mg twice daily or about 450 mg twice daily.

[0362] In some embodiments of the disclosed method, the method generates one or more of the following pharmacokinetic parameters in the plasma of the subject:

[0363] ROmax (%) is 50-100, optionally 90-100;

[0364] The average RO (%) is 50-95, optionally 65-95;

[0365] receptor expression changes max (%) is -20 to -60, optionally -35 to -60;

[0366] The mean change in receptor expression (%) was -10 to -55, optionally -25 to -55;

[0367] The steady-state ROmax (%) is 80-100;

[0368] Average RO 0-24 (hours%) is 75-90 or 50-95, optionally 65-95;

[0369] Average RO 0-12 (Hourly %) is 80-95%; and

[0370] Average RO12-24 (Hourly %) is 70-90%.

[0371] In specific embodiments of these methods, the methods include orally administering the antagonist disclosed herein, optionally compound A, at a dose of about 150 mg twice daily or about 450 mg twice daily.

[0372] In specific embodiments of the methods disclosed herein, the subject is provided with a dose or amount of an α4β7 integrin antagonist (or other agent) that does not saturate blood receptors on circulating T cells, such as α4β7 integrin receptors. Therefore, the dose or amount is one that produces a subsaturated blood receptor occupancy (RO%). In specific embodiments, the dose produces an RO% of less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%. In some embodiments, the RO% is less than 50% or less than 40%. The RO% can be measured at the drug level or at the maximum RO%. In some embodiments, the maximum RO is determined approximately four hours after administration, while the trough level appears approximately 24 hours after administration. In some embodiments, the method is practiced using peptide dimer compounds disclosed herein, such as compound A or compound B. In specific embodiments, the dose is administered orally or topically, such as rectally. In specific embodiments, this dose is administered to the subject once or twice daily.

[0373] In some embodiments of the methods disclosed herein, a dose or amount of an α4β7 integrin antagonist (or other agent) is administered to the subject to achieve high antagonist levels and / or T-cell α4β7 occupancy in gastrointestinal tissue. In specific embodiments, the dose results in T-cell α4β7 occupancy in the GI mucosa of at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, or at least 30%. In some embodiments, the methods are practiced using peptide dimer compounds disclosed herein, such as compound A or compound B. In specific embodiments, the dose is administered orally or topically, such as rectally. In specific embodiments, this dose is administered to the subject once or twice daily.

[0374] In some embodiments of the methods disclosed herein, a dose or amount of an α4β7 integrin antagonist (or other agent) is provided to the subject to achieve a blood RO% / Pierre node (or other GI tissue) RO% ratio of less than 1.0, less than 0.9, less than 0.8, less than 0.7, less than 0.6, or less than 0.5.

[0375] In a particular embodiment, the subject is provided with a dose or amount of about any one of the following: 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12.5 mg, 25.0 mg, 37.5 mg, 50.0 mg, 62.5 mg, 75 mg, 87.5 mg, 100.0 mg, 112.5 mg, 125.0 mg, 137.5 mg, 150.0 mg, 162.5 mg, 175 mg, 187.5 mg, 200.0 mg, or 212.5 mg. 225.0mg, 237.5mg, 250.0mg, 262.5mg, 275mg, 287.5mg, 300.0mg, 312.5mg, 325.0mg, 337.5mg, 350.0mg, 362.5mg, 375mg, 387.5mg, 400.0mg, 412.5mg, 425.0mg, 437.5mg, 450.0mg, 462.5mg, 475mg, 487.5mg, or 500.0mg. In some embodiments, the subject is provided with a dose of about any one of the following: 12.5 mg, 25.0 mg, 37.5 mg, 50.0 mg, 62.5 mg, 75 mg, 87.5 mg, 100.0 mg, 112.5 mg, 125.0 mg, 137.5 mg, 150.0 mg, 162.5 mg, 175 mg, 187.5 mg, 200.0 mg, 212.5 mg, 225.0 mg, 237.5 mg, 250.0 mg, 262.5 mg, 275 mg, 287.5 mg, 300.0 mg, 350.0 mg, 400.0 mg, 450.0 mg, or 500.0 mg. In some embodiments, the subject is given a dose of about any one of the following: 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, or 130 mg. In some embodiments, the subject is given a dose of about any one of the following: 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, or 115 mg. In some embodiments, the subject is given a dose of about any one of the following: 95 mg, 100 mg, or 105 mg. In some embodiments, the subject is given a dose of about 100 mg. In some embodiments, the subject is optionally given a dose ranging from about 100 mg to about 500 mg once daily or twice daily. In some embodiments, the subject is given a dose ranging from about 200 mg to about 1000 mg, optionally taken as a once daily dose or as divided doses (e.g., half the dose) twice daily.In some embodiments, a dose ranging from about 100 mg to about 1500 mg is administered to the subject daily, optionally as a once-daily dose or as a divided dose (e.g., half the dose) twice daily. In some embodiments, a dose ranging from about 100 mg to about 1500 mg is administered to the subject once or twice daily. In some embodiments, a dose of about any of the following is administered to the subject once or twice daily: 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 250 mg, 400 mg, 450 mg, or 500 mg. In some embodiments, a dose of about any of the following is administered to the subject daily: 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg, optionally as a once-daily dose or as a divided dose (e.g., half the dose) twice daily. In some embodiments, about 450 mg or about 150 mg is optionally administered twice daily. In certain embodiments, a dose of approximately any of these doses is administered to the subject twice daily, optionally orally. In certain embodiments, this dose is administered to the subject once or twice daily. In some embodiments, this dose is split, and half is administered twice daily. In some embodiments, the dose comprises a peptide dimer compound disclosed herein, such as compound A or compound B. In certain embodiments, the dose is administered orally or topically, such as rectally, optionally to treat IBD, such as ulcerative colitis.

[0376] In specific embodiments of any of the methods disclosed herein, optionally, a dose or amount of about any one of the following is administered to the subject twice daily: 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12.5 mg, 25.0 mg, 37.5 mg, 50.0 mg, 62.5 mg, 75 mg, 87.5 mg, or 100.0 mg. In some embodiments, a dose of about the following is administered to the subject: 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12.5 mg, 25.0 mg, or 37.5 mg. In some embodiments, a dose ranging from about 5 mg to about 130 mg is administered to the subject. In some embodiments, a dose ranging from about 5 mg to about 50 mg is administered to the subject. In some embodiments, a dose ranging from about 5 mg to about 12.5 mg is administered to the subject. In some embodiments, a dose of about 8 mg is administered to the subject. In some embodiments, a dose of about 150 mg twice daily or a dose of about 450 mg twice daily is administered to the subject. In certain embodiments, any of these doses is administered to the subject twice daily, optionally orally. In certain embodiments, any of these doses is administered to the subject once or twice daily. In certain embodiments, the dose is administered twice daily. In some embodiments, a dose of about 8 mg is administered to the subject. In some embodiments, a dose of about 150 mg twice daily or a dose of about 450 mg twice daily is administered to the subject. In some embodiments, the dose comprises a peptide dimer compound disclosed herein, such as compound A or compound B. In certain embodiments, the dose is administered orally or topically, such as rectally, for example by suppository. In some embodiments, a dose of about 150 mg or about 450 mg twice daily of compound A or compound B is administered orally to the subject twice daily, optionally for the treatment of IBD, such as ulcerative colitis.

[0377] In some embodiments of the methods disclosed herein, the methods are used to treat an individual or subject suffering from an inflammatory disease or condition. In a particular embodiment, the condition is an inflammatory condition of the gastrointestinal system. In some embodiments, a dose or amount of an α4β7 integrin antagonist is administered to the subject to produce a subsaturated blood receptor occupancy (RO%). In some embodiments, the methods are practiced using peptide dimer compounds disclosed herein, such as compound A or compound B. In a particular embodiment, the dose is administered orally or topically, for example, rectally. In a particular embodiment, this dose is administered to the subject once or twice daily.

[0378] In some embodiments, the disease or condition is selected from the group consisting of: inflammatory bowel disease (IBD), adult IBD, pediatric IBD, adolescent IBD, ulcerative colitis, Crohn's disease, celiac disease (non-tropical stomatitis), enteropathy associated with seroreactive arthropathy, microscopic colitis, collagenous colitis, eosinophilic gastroenteritis, radiation therapy, chemotherapy, pouchitis following rectocele and ileoanal anastomosis, gastrointestinal cancer, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, pericholangitis, chronic bronchitis, chronic sinusitis, asthma, primary sclerosing cholangitis, HIV infection, eosinophilic asthma, eosinophilic esophagitis, gastritis, colitis, microscopic colitis, and graft-versus-host disease (GVDH). In a particular embodiment, the disease or condition is IBD. In some embodiments, the IBD is ulcerative colitis. In some embodiments, the IBD is Crohn's disease. In some embodiments, compound A or compound B is administered orally to a subject to treat ulcerative colitis or Crohn's disease.

[0379] In some embodiments, this disclosure provides a method of treating an IBD in a subject of need, comprising orally administering to the subject a peptide dimer compound disclosed herein, such as compound A or compound B, wherein the compound is administered at a dose that produces a subsaturated blood receptor occupancy, such as less than 50% RO. In some embodiments, the IBD is ulcerative colitis or Crohn's disease. In specific embodiments, the subject is provided with a dose or amount of about any of the following: 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12.5 mg, 25.0 mg, 37.5 mg, 50.0 mg, 62.5 mg, 75 mg, 87.5 mg, 100.0 mg, 112.5 mg, 125.0 mg, 137.5 mg, 150.0 mg, 162.5 mg, 175 mg, 187.5 mg, 200.0 mg, or 212.5 mg. 225.0mg, 237.5mg, 250.0mg, 262.5mg, 275mg, 287.5mg, 300.0mg, 312.5mg, 325.0mg, 337.5mg, 350.0mg, 362.5mg, 375mg, 387.5mg, 400.0mg, 412.5mg, 425.0mg, 437.5mg, 450.0mg, 462.5mg, 475mg, 487.5mg, or 500.0mg. In some embodiments, the subject is provided with a dose of about any one of the following: 12.5 mg, 25.0 mg, 37.5 mg, 50.0 mg, 62.5 mg, 75 mg, 87.5 mg, 100.0 mg, 112.5 mg, 125.0 mg, 137.5 mg, 150.0 mg, 162.5 mg, 175 mg, 187.5 mg, 200.0 mg, 212.5 mg, 225.0 mg, 237.5 mg, 250.0 mg, 262.5 mg, 275 mg, 287.5 mg, 300.0 mg, 350.0 mg, 400.0 mg, 450.0 mg, or 500.0 mg. In some embodiments, the subject is given a dose of about any one of the following: 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, or 130 mg. In some embodiments, the subject is given a dose of about any one of the following: 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, or 115 mg. In some embodiments, the subject is given a dose of about any one of the following: 95 mg, 100 mg, or 105 mg. In some embodiments, the subject is given a dose of about 100 mg.In some embodiments, a dose ranging from about 100 mg to about 500 mg is optionally administered to the subject once or twice daily. In some embodiments, a dose ranging from about 200 mg to about 1000 mg is administered to the subject, optionally as a once-daily dose or as a divided dose (e.g., half the dose) twice daily. In some embodiments, a dose ranging from about 100 mg to about 1500 mg is administered to the subject daily, optionally as a once-daily dose or as a divided dose (e.g., half the dose) twice daily. In some embodiments, a dose ranging from about 100 mg to about 1500 mg is administered to the subject once or twice daily. In some embodiments, a dose of about any one of the following is administered to the subject once or twice daily: 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 250 mg, 400 mg, 450 mg, or 500 mg. In some embodiments, a dose of about any one of the following is provided to the subject daily: 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg, optionally taken once daily or in divided doses (e.g., half the dose) twice daily. In some embodiments, about 450 mg or about 150 mg is optionally provided twice daily. In some embodiments, a dose of compound A or compound B of about 150 mg twice daily or about 450 mg twice daily is provided to the subject to treat ulcerative colitis (UC) or Crohn's disease. In some embodiments, the method is used to treat ulcerative colitis. In certain embodiments, the subject has moderate to severe active UC. In some embodiments, the subject has a biopsy-confirmed diagnosis of UC. In some embodiments, the subject meets one or more (or all) of the inclusion criteria disclosed in the examples and does not meet one or more (or any) of the exclusion criteria disclosed in the examples.

[0380] In some embodiments, this disclosure provides a method of treating IBD (e.g., ulcerative colitis or Crohn's disease) in a subject of need, the method comprising orally administering to the subject a peptide dimer compound disclosed herein, such as compound A or compound B, wherein the compound is administered at a dose that produces plasma in the subject that satisfies one or more of the following pharmacokinetic parameters:

[0381] Cmax (ng / mL) is 1-25, optionally 4-12;

[0382] Tmax (hours) is 1-5, and can be selected as 2-4;

[0383] AUC t(nanogram-hours / mL) is 10-250, optionally 50-150;

[0384] AUC inf (nanogram-hours / mL) is 10-300, optionally 30-250;

[0385] t 1 / 2 (Hours) is 3-10, and optional locations are 4-10;

[0386] AUC tau (nanogram-hours / mL) is 30-130;

[0387] Ctrough (ng / mL) is 1-5;

[0388] The cumulative Cmax (ng.mL) was 0.5–2.5, optionally 2–3; and

[0389] Cumulative AUC t (Ng·h / mL) is 0.5-3.0.

[0390] In specific embodiments of these methods, the IBD is ulcerative colitis or Crohn's disease. In specific embodiments, the pharmacokinetic parameters are met within 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, or 12 hours after administration. In specific embodiments, the pharmacokinetic parameters are maintained for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 8 hours, or at least 12 hours after administration.

[0391] In some embodiments, this disclosure provides a method of treating IBD in a subject of need, the method comprising orally administering to the subject a peptide dimer compound disclosed herein, such as compound A or compound B, wherein the compound is administered at a dose that produces one or more of the following pharmacodynamic parameters in the subject's plasma:

[0392] ROmax (%) is 50-100, optionally 90-100;

[0393] The average RO (%) is 50-95, optionally 65-95;

[0394] receptor expression changes max (%) is -20 to -60, optionally -35 to -60;

[0395] The mean change in receptor expression (%) was -10 to -55, optionally -25 to -55;

[0396] The steady-state ROmax (%) is 80-100;

[0397] Average RO 0-24 (Hourly %) is 75-90;

[0398] Average RO 0-12 (Hourly %) is 80-95%; and

[0399] Average RO 12-24 (Hourly %) is 70-90%.

[0400] In specific embodiments of these methods, the IBD is ulcerative colitis or Crohn's disease. In specific embodiments, the pharmacodynamic parameters are met within 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, or 12 hours after administration. In specific embodiments, the pharmacodynamic parameters are maintained for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 8 hours, or at least 12 hours after administration.

[0401] In some embodiments, the methods disclosed herein reduce the activity (partially or completely) of α4β7 in a subject. In some embodiments, the methods reduce the proliferation of T cells including α4β7 integrin, for example, the proliferation of T cells present in the subject's gastrointestinal tissue, such as the gastrointestinal mucosa. In other embodiments, the methods inhibit the production or release of cytokines by the subject's T cells, such as β7+ T cells in the subject's gastrointestinal tissue. In specific embodiments, the methods reduce the production or release of any of the cytokines disclosed in the accompanying drawings, such as IFNγ, interleukin-6 (IL-6), IL-8, IL-12 / 23p40, IL-15, IL-16, IL-13, vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor α (TNFα), or tumor necrosis factor β (TNFβ). In some embodiments, the methods disclosed herein inhibit the production or release of cytokines by T cells, the release of which is promoted by binding to the subject's mucosal vascular addressing cell adhesion molecule 1 (MAdCAM1), for example, in gastrointestinal tissues, such as the gastrointestinal mucosa. In certain embodiments, the T cells are CD45RO-primary or CD45RO+ memory T cells. In some embodiments, the T cells are β7 + .

[0402] In other related embodiments, the present invention includes a method for treating a subject, such as a mammal or human, suffering from a symptom associated with the biological function of α4β7, the method comprising providing or administering to the subject an amount of the peptide molecule described herein sufficient to inhibit (partially or completely) the biological function of α4β7 in tissues expressing MAdCAM1, such as gastrointestinal tissue, like the gastrointestinal mucosa. In a particular embodiment, the subject is provided with an effective amount of peptide monomer or peptide dimer sufficient to at least partially inhibit the biological function of α4β7 in tissues expressing MAdCAM1. In some embodiments, the symptom is inflammatory bowel disease.

[0403] In another embodiment, the invention includes a method for treating or preventing a disease or symptom in a subject of need, the method comprising providing or administering to the subject, for example, a mammal, an effective amount of the peptide dimer or peptide monomer described herein, wherein the disease or symptom is selected from the group consisting of: inflammatory bowel disease (IBD) (including adult IBD, pediatric IBD, and adolescent IBD), ulcerative colitis, Crohn's disease, celiac disease (non-tropical stomatitis), enteropathy associated with seroreactive arthropathy, and microscopic colon. Inflammation, collagenous colitis, eosinophilic gastroenteritis, pouchitis following radiotherapy, chemotherapy, rectocolectomy and ileoanal anastomosis, gastrointestinal cancer, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, pericholangitis, chronic bronchitis, chronic sinusitis, asthma, primary sclerosing cholangitis, HIV infection, eosinophilic asthma, eosinophilic esophagitis, gastritis, colitis, microscopic colitis, and graft-versus-host disease (GVDH) (including intestinal GVDH). In any specific embodiment of the treatment methods described herein, the subject has been diagnosed with or is considered at risk of developing one of these diseases or conditions.

[0404] In any specific embodiment of the treatment methods described herein, a peptide molecule (or a pharmaceutical composition comprising the peptide molecule) is administered to an individual in a manner selected from the group consisting of: oral, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intrathecal, inhalation, vaporization, nebulization, sublingual, oral, parenteral, rectal, vaginal, and local administration.

[0405] In certain embodiments, this disclosure provides unit dosage forms of the peptide dimer compounds disclosed herein, said unit dosage forms comprising about any one of the following: 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12.5 mg, 25.0 mg, 37.5 mg, 50.0 mg, 62.5 mg, 75 mg, 87.5 mg, 100.0 mg, 112.5 mg, 125.0 mg, 137.5 mg, 150.0 mg, 162.5 mg, 175 mg, 187.5 mg, 20 mg, etc. 0.0mg, 212.5mg, 225.0mg, 237.5mg, 250.0mg, 262.5mg, 275mg, 287.5mg, 300.0mg, 312.5mg, 325.0mg, 337.5mg, 350.0mg, 362.5mg, 375mg, 387.5mg, 400.0mg, 412.5mg, 425.0mg, 437.5mg, 450.0mg, 462.5mg, 475mg, 487.5mg, or 500.0mg. In some embodiments, the unit dosage form includes about any one of the following: 12.5 mg, 25.0 mg, 37.5 mg, 50.0 mg, 62.5 mg, 75 mg, 87.5 mg, 100.0 mg, 112.5 mg, 125.0 mg, 137.5 mg, 150.0 mg, 162.5 mg, 175 mg, 187.5 mg, 200.0 mg, 212.5 mg, 225.0 mg, 237.5 mg, 250.0 mg, 262.5 mg, 275 mg, 287.5 mg, 300.0 mg, 350.0 mg, 400.0 mg, 450.0 mg, or 500.0 mg. In some embodiments, the unit dosage form includes about any one of the following: 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, or 130 mg. In some embodiments, the unit dosage form includes about any one of the following: 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, or 115 mg. In some embodiments, the unit dosage form includes about any one of the following: 95 mg, 100 mg, or 105 mg. In some embodiments, the unit dosage form includes about 100 mg. In some embodiments, the unit dosage form includes about 100 mg to 500 mg. In some embodiments, the unit dosage form includes about any one of the following unit dosage forms: 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 250 mg, 400 mg, 450 mg, or 500 mg.In some embodiments, the unit dosage form comprises about 450 mg or about 150 mg. In a particular embodiment, the unit dosage form comprises a pharmaceutical composition comprising a peptide dimer compound, such as any of the peptide dimer compounds disclosed herein. In a particular embodiment, the unit dosage form is formulated for oral administration, for example, as a tablet. In some embodiments, the unit dosage form is formulated for rectal administration, for example, as a suppository. In some embodiments, the unit dosage form comprises about 450 mg or about 150 mg of compound A or compound B (or a pharmaceutically acceptable salt thereof). In a particular embodiment, the unit dosage form comprises a pharmaceutical composition comprising a peptide dimer compound, such as any of the peptide dimer compounds disclosed herein.

[0406] In certain embodiments, the peptide molecules of the present invention are present in a pharmaceutical composition further comprising one or more pharmaceutically acceptable diluents, carriers, or excipients. In certain embodiments, the peptide molecules of the present invention are formulated as liquids or solids. In certain embodiments, the peptide molecules of the present invention are formulated as tablets or capsules or as liquid suspensions. Some embodiments of the present invention further provide a method for treating an individual using the α4β7 integrin antagonist peptide molecules of the present invention suspended in a sustained-release matrix. As used herein, the sustained-release matrix is ​​a matrix made of a material (typically a polymer) that can be hydrolyzed by enzymes or acids or degraded by dissolution. Once inserted into the body, the matrix is ​​subjected to the action of enzymes and body fluids. The sustained-release matrix is ​​ideally selected from biocompatible materials such as liposomes, polylactide (polylactic acid), polyglycolic acid (polymer of glycolic acid), polylactide-co-glycolic acid (copolymer of lactic acid and glycolic acid), polyanhydrides, poly(ortho-esters), peptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinylpropylene, polyvinylpyrrolidone, and siloxanes. A specific biodegradable matrix is ​​one of polylactide, polyglycolic acid, or polylactide-co-glycolic acid (copolymer of lactic acid and glycolic acid).

[0407] In some aspects, the present invention provides a pharmaceutical composition for oral delivery. Various embodiments and peptide compositions of the present invention can be prepared for oral administration according to any of the methods, techniques, and / or delivery media described herein. Furthermore, those skilled in the art will understand that the peptide compositions of the present invention can be modified or integrated into systems or delivery media not disclosed herein but well known in the art and suitable for the oral delivery of small peptide molecules.

[0408] Oral dosage forms or unit doses compatible with the use of the peptides of the present invention may comprise a mixture of peptide-active pharmaceutical components and non-pharmaceutical components or excipients, as well as other non-reusable materials that may be considered as ingredients or packaging. Oral compositions may comprise at least one of liquid, solid, and semi-solid dosage forms. In some embodiments, an oral dosage form comprising an effective amount of the peptide molecules described herein is provided, wherein the dosage form comprises at least one of pills, tablets, capsules, gels, pastes, beverages, and syrups. In some instances, an oral dosage form is provided that is designed and configured to achieve a delayed release of thioether peptide molecules in the small intestine of a subject.

[0409] In one embodiment, an oral pharmaceutical composition comprising the peptide of the present invention includes an enteric coating designed to delay the release of the peptide molecule in the small intestine. In some instances, it is preferred that the pharmaceutical composition of the present invention includes an enteric coating soluble in gastric juice at a pH of about 5.0 or higher. In at least one embodiment, a pharmaceutical composition is provided comprising an enteric coating comprising a polymer having a dissociable carboxyl group, such as a cellulose derivative, including hydroxypropyl methylcellulose phthalate, cellulose acetate phthalate, and cellulose trimellitate, as well as similar derivatives of cellulose and other carbohydrate polymers.

[0410] In one embodiment, a pharmaceutical composition comprising the peptide molecules described herein is provided in an enteric coating designed to protect and release the pharmaceutical composition in a controlled manner within the lower gastrointestinal system of a subject, avoiding systemic side effects. In addition to enteric coating, the peptide molecules of the present invention can be encapsulated, coated, conjugated, or otherwise associated with any compatible oral drug delivery system or component. For example, in some embodiments, the peptide molecules of the present invention are provided in a lipid carrier system comprising at least one of polymeric hydrogels, nanoparticles, microspheres, micelles, and other lipid systems.

[0411] To overcome peptide degradation in the small intestine, some embodiments of the present invention include a hydrogel polymer carrier system containing peptide molecules according to the invention, whereby the hydrogel polymer protects the peptide from proteolytic hydrolysis in the small intestine. The peptide molecules of the present invention can be further formulated for compatibility with carrier systems designed to increase dissolution kinetics and enhance intestinal absorption of the peptide. These methods include the use of liposomes, micelles, and nanoparticles to increase the GI channel permeability of the peptide.

[0412] Various bioresponsive systems can also be combined with one or more thioether peptide molecules of the present invention to provide pharmaceutical agents for oral delivery. In some embodiments, the peptide molecules of the present invention are combined with mucosal adhesion polymers such as hydrogels and those having hydrogen-bonding groups (e.g., PEG, poly(methacrylic) acid [PMAA], cellulose, etc.). Bioresponsive systems such as chitosan and alginate are used in combination to provide therapeutic agents for oral administration. Other embodiments include methods for optimizing or prolonging the drug residence time of peptide molecules disclosed herein, wherein the surface of said peptide molecules is modified to include mucosal adhesive properties via hydrogen bonds, polymers with linked mucins, and / or hydrophobic interactions. According to the desired features of the invention, these modified peptide molecules can be shown to increase the drug residence time in a subject. Furthermore, targeted mucosal adhesion systems can specifically bind to receptors on the surface of intestinal epithelial cells and M cells, thereby further increasing the uptake of peptide-containing particles.

[0413] Other embodiments include methods for oral delivery of the peptide molecules described herein, wherein the peptide molecules are used in combination with a permeation enhancer that promotes peptide transport across the intestinal mucosa by increasing paracellular or transcellular permeability. For example, in one embodiment, a permeation enhancer is combined with the peptide molecules described herein, wherein the permeation enhancer comprises at least one of long-chain fatty acids, bile salts, amphiphilic surfactants, and chelating agents. In one embodiment, a permeation enhancer comprising sodium N-[(hydroxybenzoyl)amino]octanoate is used to form a weak non-covalent association with the peptide molecules of the present invention, wherein the permeation enhancer facilitates membrane transport and further dissociation once in the bloodstream. In other embodiments, the peptide molecules are conjugated with oligoarginine, thereby increasing peptide permeability into cells of various cell types. Further, in at least one embodiment, a non-covalent bond is provided between the peptide molecules described herein and a permeation enhancer selected from the group consisting of cyclodextrins (CDs) and dendritic compounds, wherein the permeation enhancer reduces peptide aggregation and increases the stability and solubility of the peptide molecules.

[0414] When used in at least one of the therapeutic or delivery systems described herein, a therapeutically effective amount of one of the peptide molecules of the present invention may be used in its pure form or, where such a form is available, in the form of a pharmaceutically acceptable salt. As used herein, a “therapeutically effective amount” of the compounds of the present invention means a sufficient amount of peptide molecules for treating integrin-related diseases (e.g., for reducing inflammation associated with IBD) in a benefit / risk ratio suitable for any medical treatment. However, it should be understood that the total daily dose of the compounds and compositions of the present invention will be determined by the attending physician within the bounds of reasonable medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a number of factors, including: a) the condition being treated and the severity of said condition; b) the activity of the specific compound used; c) the specific composition used by the patient, age, weight, general health condition, sex, and diet; d) the timing, route of administration, and excretion rate of the specific compound used; e) the duration of treatment; f) the drugs used in combination with or concurrently with the specific compound used, and similar factors well known in the medical field.

[0415] Alternatively, the compounds of the present invention can be administered as pharmaceutical compositions comprising the peptide molecule of interest in combination with one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable carriers or excipients refer to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid. The compositions can be administered parenterally, intracisional, intravaginally, intraperitoneally, rectally, topically (e.g., via powder, ointment, drops, suppositories, or transdermal patches), rectally, or orally. As used herein, the term “parenteral” refers to a mode of administration that includes intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, intradermal, and intra-articular injection and infusion.

[0416] The composition for rectal or vaginal application is preferably a suppository, which can be prepared by mixing the compound of the invention with a suitable non-irritating excipient or carrier (such as cocoa butter, polyethylene glycol or suppository wax), which is solid at room temperature but liquid at body temperature and thus can melt in the rectal or vaginal cavity to release the active compound.

[0417] The total daily dose of the composition of the invention administered to a human or other mammalian host in a single dose or in multiple doses may be, for example, from 0.0001 mg / kg body weight to 300 mg / kg body weight per day, and more typically from 1 mg / kg body weight to 300 mg / kg body weight per day.

[0418] Example

[0419] Example 1

[0420] Compound A blocks MADCAM1-mediated CD4+ T cell proliferation.

[0421] The oral gastrointestinal (GI)-restricting peptide antagonist, α4β7 integrin compound A, is being developed for the treatment of inflammatory bowel disease (IBD). Blocking the binding of α4β7 to the mucosal addressing cell adhesion molecule 1 (MAdCAM1) is thought to treat IBD by preventing the extravascular migration of blood T cells to the inflamed GI mucosa. The following experiments were conducted to further explore the mechanism by which compound A reduces GI inflammation. Specifically, by evaluating the inhibition of MAdCAM1-mediated CD4+ by compound A. + The potential to assess the local GI function of α4β7 by examining the ability of T cells to proliferate and produce cytokines.

[0422] Compound A:

[0423]

[0424] ((2-benzyl)-(N-Me-R)-Ser-Asp-Thr-Leu-Pen-(Phe(4-tBu))-(β-homo-Glu)-(D-Lys)-OH)2(SEQ ID NO:5) and linker-DIG diethylene glycol.

[0425] PBMCs were purified from healthy human donors and enriched with CD4+ T cells. Primary CD4+ T cells were then processed. + T cells were fluorescently labeled and incubated alone with plate-bound anti-CD3, or with MAdCAM1 for three days with or without the following inhibitors (or negative controls): inactive analog compound A (1 μM) or vedolizumab (500 ng / mL) as a negative control (1 μM). Phenotypic analysis, T helper (Th) subset distribution, and RO% were performed on freshly stained live samples by flow cytometry.

[0426] After 3 days of incubation, the combination of MAdCAM1 and anti-CD3 significantly enhanced CD4 compared to anti-CD3 alone. + T cell proliferation (n=7, 12%-87%) Figure 1 Compound A completely eliminated MAdCAM1-mediated proliferation. Figure 1 The inhibition level was similar to that of vedolizumab. Figure 1 No blocking was observed with the inactive analogue (negative control; NEG), indicating a dependence on the binding of compound A to α4β7. Inhibition by compound A depended on the activity of compound A. Inhibition by compound A was concentration-dependent. The mean IC50 from four independent human donors was 4.4 nM (Table 4).

[0427] Table 4. IC50 from four donors

[0428] N=3 donors IC50(nM) Donor 7 1.6 Donor 8 3.7 Donor 9 5.2 Donor 11 8.0 average value 3.5 Standard deviation 1.8

[0429] Immunophenotypic analysis revealed that proliferation occurred on CD45RO - Original and CD45RO + Memory T cells, and the transformation of primordial T cells into memory cell phenotypes ( Figure 2 Proliferation is limited to β7. + The population, in which continuous cycles of proliferation show increased β7 expression ( Figure 3 A). In the presence of compound A, β7 is expressed on the surface of undivided CD4 cells. + Decreased in T cells, indicating internalization via compound A ( Figure 3 B and Figure 4 (Tested in 5 donors). In proliferating memory T cells, the percentage of the Th1 subset producing IFNγ was higher than that of the Th17 subset producing IL-17A and the Th2 subset producing IL-4 (Table 5).

[0430] Table 5. Characteristics of proliferating CD4+ T cells

[0431]

[0432] α4β7-MAdCAM1 interaction promotes β7 + CD4 + T cell proliferation and cytokine release may lead to a chronic inflammatory response in the diseased gut of IBD patients, independent of T cell transport. Compound A supports the potential therapeutic advantage of oral GI-restricted approaches by inhibiting MAdCAM1-mediated signaling via α4β7, where compound A is locally delivered and directly blocks α4β7 function in the GI.

[0433] Example 2

[0434] Compound A blocks MADCAM1-mediated cytokine production.

[0435] The following experiments were conducted to further explore the mechanism by which compound A reduces GI inflammation. Specifically, cytokine analysis was performed on T cells isolated from normal healthy donors.

[0436] PBMCs were purified from three healthy human donors (donors 7, 10, and 11) and enriched with CD4+ T cells. Primary CD4+ T cells were then used for further purification. +T cells were fluorescently labeled and incubated alone with plate-bound anti-CD3, incubated with plate-bound anti-CD3 and MAdCAM1 together, or incubated with plate-bound anti-CD3, MAdCAM1, and various amounts of compound A. Cytokine levels in the supernatant of anti-CD3 alone and anti-CD3+MAdCAM1 in the presence of different concentrations of compound A were quantified using multiplex assays on the MSD or Luminex platform.

[0437] Multiplex analysis identified several cytokines, including IFNγ, IL-5, IL-6, IL-10, IL-13, GM-CSF, and TNFα, the release of which was promoted by MAdCAM1. Figure 5 AC and 6A-C). Compound A inhibits MAdCAM1-mediated cytokine production in a concentration-dependent manner. The concentration-dependent and complete inhibition of specific MAdCAM1-mediated cytokine production by compound A are demonstrated in... Figure 5 In AC and 6A-C, the α4β7-MAdCAM1 interaction promotes β7... + CD4 + T cell proliferation and cytokine release may lead to a chronic inflammatory response in the diseased gut of IBD patients, independent of T cell transport. Compound A supports the therapeutic advantage of oral GI-restricted approaches by inhibiting MAdCAM1-mediated signaling via α4β7, where compound A is locally delivered and directly blocks α4β7 function in the GI.

[0438] Example 3

[0439] Receptor occupancy in mice

[0440] The following experiments were performed to examine receptor occupancy in whole blood and Pierre nodes of mice administered compound A analogues and compound B.

[0441] Compound B:

[0442]

[0443] (Ac-Pen-(N-Me-Arg)-Ser-Asp-Thr-Leu-Pen-(Phe(4-tBu))-(β-homo-Glu)-(D-Lys)-NH2)2(SEQ ID NO:6) and linker =DIG (diethylene glycol).

[0444] Three groups of female C57BL / 6 mice (N=6 per group) were orally administered a mordant (Group 1), compound B (3 mg / kg, PO, QD; Group 2), or compound B (30 mg / kg, PO, QD; Group 3). One hour after administration, the mice were euthanized and whole blood / plasma and Peyer's nodes were collected. Peyer's nodes were dispersed in 1 mL of RPMI medium containing 2% FBS, without washing. Single-cell suspensions of whole blood and Peyer's nodes (100 μL out of a total of 1 mL) were submitted for flow cytometry to determine α4β7 receptor occupancy. RO% = (1 - (median positive percentage of test sample / positive percentage of mordant control)) * 100. Drug exposure in plasma and Peyer's nodes dispersed in single-cell suspensions (1 mL out of a total of 500 μL) was also analyzed.

[0445] Compared with whole blood in both dosage groups, the receptor occupancy in the Peyer's node was significantly higher. Figure 7 The levels of receptor occupancy in whole blood or Pierre nodes were comparable across the dose groups. At both 3 mg / kg and 30 mg / kg doses ( Figure 8 (Top) Competitive (100%) receptor occupancy exists in the Pierre nodes of some animals. Figure 8 The bottom shows the RO% for various doses of compound B. Figure 9 The data shows PK and PD data, including the dosage of compound B.

[0446] Similar experiments were conducted using compound A. Receptor occupancy in the Peyer's node was significantly higher than in whole blood in both dosage groups. At a dose of 30 mg / kg, receptor occupancy in both whole blood (P<0.01) and Peyer's node (P<0.001) was significantly higher than at a dose of 3 mg / kg. Figure 25 The same effect was observed using compound B, although in smaller quantities, possibly because compound A has greater activity than compound B, but at the same dose. There was also a significant dose-dependent increase in the concentration of compound A in both plasma and Pierre's nodes. Figure 26 At both dose levels, the concentration of compound A in the Peyer's knot was significantly higher than that in plasma. Figure 27 As shown, receptor occupancy in whole blood and Peyer's nodes increased in a dose-dependent manner in animals treated with compound A. In animals at a dose of 30 mg / kg, complete (100%) receptor occupancy was observed in Peyer's nodes.

[0447] Example 4

[0448] Compound A tissue exposure in mice

[0449] This study was conducted to determine the plasma, Pierre's nodes (PP), mesenteric lymph nodes (MLN), small intestine, and colon tissue exposure of compound A after administration of PO to healthy C57BL / 6 female mice.

[0450] Twelve untreated female C57BL / 6 mice were assigned to this study. Animals were fasted overnight and administered a single dose of compound A at a dose-volume of 10 mL / kg via force-feed (PO). At 1 hour, 3 hours, and 6 hours post-administration, four mice / time points were subjected to terminal exsanguination and euthanasia; Peyer's nodes (PP), mesenteric lymph nodes (MLN), small intestine, and colon were collected from each animal. Blood was processed into plasma; plasma and tissue samples were submitted for pharmacokinetic (PK) analysis of compound A levels using qualified liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0451] The concentration of compound A in plasma and tissue was analyzed using a qualified liquid chromatography-tandem mass spectrometry (LC-MS / MS) method. Processed plasma and tissue samples were analyzed on an AB / MDS Sciex API 4000 mass spectrometer. Cations were monitored in multiple reaction monitoring (MRM) mode. Quantification was performed by peak area ratio.

[0452] PK data analysis was performed using non-compartmental analysis (NCA) on Phoenix WinNonlin 8.1 (Certara USA Inc., USA). For pharmacokinetic analysis, all concentration values ​​below the limit of quantitation were considered zero. The maximum concentration (Cmax) and the apparent time to reach Cmax (tmax) were obtained by observation. The area under the concentration versus time curve (AUC) was obtained using a linear trapezoidal method. If the value is greater than one, all concentration data and PK parameters are reported to a maximum of three significant figures, and if the value is less than one, it is reported to a maximum of three decimal places. Time parameters are reported to a maximum of two decimal places. Concentration data were plotted using Excel (Microsoft).

[0453] Figure 10 The average plasma and tissue concentrations of compound A in each animal were plotted. Figure 11The obtained pharmacokinetic parameters are provided. Peak compound A exposure was observed at 1 hour in the MLN, PP, and small intestine, 3 hours in plasma, and 6 hours in the colon following a single PO administration of 30 mg / kg. The mean Cmax value was highest in the small intestine (13300 ng / g), and approximately half that was observed in the PP and colon. These gastrointestinal levels were significantly higher (100-fold or more) than those in plasma (19.0 ng / mL) and MLN (56.8 ng / g). Similarly, the mean AUC values ​​in the small intestine, PP, and colon (48600 ng / g, 37900 ng / g, and 15700 ng / g, respectively) were significantly higher (60-fold or more) than those in plasma (95.4 ng / mL) and MLN (226 ng / g). These results suggest that compound A has limited plasma and lymph node exposure when administered PO in otherwise healthy female mice. Dosage analysis (data not shown) indicates that the administered solution is 97.6% of the nominal concentration.

[0454] Example 5

[0455] Compound A inhibits intestinal homing of cultured T cells.

[0456] T cells cultured in the presence of all-trans retinoic acid (ATRA) upregulated the intestinal homing receptor CCR9, integrin α4 (α4), and integrin β7 (β7), and preferentially homed to intestinal tissues (laminar propria of the ileum and Pierre's nodes). The aim of this study was to analyze the intestinal homing of T cells cultured in the presence of compound A.

[0457] Purified CD3+ cells were isolated from B6.SJL (CD45.1+) donor mice and cultured in the presence of anti-CD3 / anti-CD28 beads and IL-2 to induce T cell activation and proliferation. Compound A and / or ATRA were added to some culture conditions. To track cells in vivo, ATRA- and ATRA+ cells were labeled with CMFDA and CTFR, respectively. These labeled cells were then co-injected into C57BL / 6 (CD45.2+) recipient mice.

[0458] This study included four groups of recipient mice:

[0459] • Mediator (negative control)

[0460] • Anti-VLA-4 (treated with anti-VLA-4 in vivo, positive control)

[0461] Compound A, 100 nM (tested, in culture)

[0462] Compound A, 1000 nM (tested, in culture)

[0463] Cell homing was assessed by measuring the proportions of ATRA- and ATRA+ cells in the spleen, Peyer's node (PP), and lamina propria (LP) of recipient mice using flow cytometry.

[0464] As expected, cells cultured in the presence of ATRA+ / DMSO (“ATRA+ / DMSO cells”) had a higher proportion of cells expressing the intestinal homing receptor CCR9 and integrins α4 and β7 compared to cells cultured in the absence of ATRA (“ATRA-cells”). The proportion of integrin β7+ cells in ATRA+ / compound A cells was lower than in ATRA+ / DMSO cells. The proportion of ATRA-cells in the spleen of mediator-treated mice was greater than that of ATRA+ cells, and the proportion of ATRA+ cells in the LPs of these mice was greater than that of ATRA-cells, confirming that ATRA+ cells preferentially home to the gut, as expected for this group. The proportion of ATRA+ cells in the LPs of anti-VLA-4 treated mice was approximately 1 / 10 that of the LPs of mediator-treated mice, and the proportion of ATRA+ cells in the PPs was approximately 1 / 2 that of the PPs of mediator-treated mice. These results confirm that anti-VLA-4 treatment reduced intestinal homing of ATRA+ cells, as expected for this positive control. Compared to the mediator group, the proportion of CD45.1+ cells in the spleen and LP was significantly lower in the compound A, 1000 nM group. Furthermore, the proportion of ATRA+ cells in the LP was lower in both compound A groups compared to the mediator group, and the reduction in the 1000 nM group was close to statistically significant.

[0465] method:

[0466] Eighteen (18) B6.SJL(CD45.1+) donor mice were acclimatized for 3 to 9 weeks prior to the start of the study and were 11 to 16 weeks old at the time of culture setup (day 0). On day 0, spleen and lymph node cells were isolated from the donor mice and pooled. CD3+ cells were enriched using a STEMCELL Technologies kit catalog number 19851. The purity of the enriched cells was confirmed by flow cytometry.

[0467] Then, in the presence of anti-CD3 / CD28 beads (Dynabead, Thermo Fisher 11453D), approximately 44% of the cells were inoculated at 1.5 × 10⁻⁶. 6 / mL of culture was performed, with a cell-to-bead ratio of 1:1 (ATRA-, according to Table 4 below). The remaining cells were cultured under the same conditions, except that the cell concentration was 2×10⁶. 6 / mL, and compound A or DMSO was added to the cultures. Then all-trans retinoic acid (ATRA) was added to these cultures at a concentration of 0.1 μM. The culture conditions are summarized in Table 6 below.

[0468] Table 6 - Culture Conditions

[0469]

[0470] On day 1, IL-2 was added to all cultures to reach a concentration of 30 U / mL.

[0471] From day 2 to day 4, expand the culture as needed by adding fresh culture medium while maintaining the following concentrations:

[0472] • The IL-2 concentration used for all cultures is 30 U / mL

[0473] • The ATRA concentration used for ATRA+ cultures is 0.1 μM.

[0474] • The concentration of DMSO used for ATRA+ cultures was 0.1%.

[0475] • Compound A listed in Table 4

[0476] On day 5, cells from each culture were stained and analyzed by flow cytometry using the reagents listed in Table 7.

[0477] Table 7 - Flow cytometry kits for assessing intestinal homing receptor expression

[0478]

[0479]

[0480] Thirty-eight (38) C57BL / 6 (CD45.2+) recipient mice were acclimated for nine weeks before the start of the study (day 0) and were 16 weeks old at the time of cell transfer (day 5). On day 4, the recipient mice were assigned to the groups in a balanced manner to achieve similar average weight across the groups.

[0481] On day 5, after removing CD3 / CD28 beads from the cell culture using a magnet, ATRA+ cells were labeled with CFTR and ATRA- cells with CMFDA. Cells from each culture condition were then counted. For each group, ATRA- cells and cells from one of the ATRA+ culture conditions were mixed at a 1:1 ratio according to Table 8 below and transferred to recipient mice. Approximately 13 million cells of each type (26 million cells in total) were intravenously injected into each mouse.

[0482] Table 8 - Treatment Plan

[0483]

[0484] On day 5 prior to cell transfer, mice in group 2 were administered anti-VLA-4 once. The anti-VLA-4 (PS / 2) antibody was purchased from BioXCell and stored at -80°C until needed. The antibody was diluted to a final concentration of 1 mg / mL with sterile PBS and administered intraperitoneally at a dose of 10 mg / kg. No in vivo treatment was performed on other groups.

[0485] Twenty (20) to 22 hours after cell transfer, all mice were euthanized and their blood, spleen, Pierre nodes, and small intestine were collected. Approximately 50 μL of plasma was isolated from the blood of each mouse and stored on dry ice until further analysis.

[0486] Cells from the following tissues were isolated from each mouse for flow cytometry analysis:

[0487] ·spleen

[0488] ·Pai'er Jie

[0489] ·Laminary tract of the ileum

[0490] The isolated cells were counted and stained with anti-CD45.1 antibody and live / dead staining agent. Cells were then harvested for flow cytometry analysis, and the proportions of CD45.1+, ATRA+, and ATRA- cells in each tissue were determined.

[0491] At the end of the culture period, flow cytometry analysis showed that the proportion of ATRA+ / DMSO cells expressing the intestinal homing receptor CCR9 and integrins α4 and β7 was much smaller compared to ATRA- cells. Figure 12 and 13 These findings confirm that cells cultured in the presence of ATRA upregulate gut homing receptors as expected. The proportion of integrin β7+ cells in ATRA+ / compound A cultures was smaller compared to ATRA+ / DMSO cultures. Figure 12 Integrin α4 expression was also lower in ATRA+ / compound A cultures compared to ATRA+ / DMSO cultures, but CCR9 expression appeared to be unaffected. Figure 13 These results indicate that compound A inhibits, upregulates, or interferes with the expression of integrins β7 and α4. The results of tissue homing analysis are shown in Tables 9-13.

[0492] Table 9 - Total number of isolated cells (×10) 3 )

[0493]

[0494] *p<0.05 for mediators

[0495] Table 10 - CD45.1+ cells / 10 3 One living cell

[0496]

[0497] *p<0.05 for mediators

[0498] Table 11 - ATRA+ cells and ATRA- cells in the spleen / 10 3 One living cell

[0499]

[0500] *p<0.05 for mediators

[0501] Table 12 - ATRA+ and ATRA- cells in Pierre nodes / 10 3 One living cell

[0502]

[0503] *p<0.05 for mediators

[0504] Table 13 - ATRA+ and ATRA- cells in the ileal LP / 10 3 One living cell

[0505]

[0506] *p<0.05 for mediators

[0507] ** p<0.10

[0508] The number of cells isolated from the spleen, Peyer's nodes, and lamina propria (LP) in the mediator group was as expected (Table 9). Similarly, the proportion of CD45.1+ cells isolated from these tissues was as expected for this model (Table 10).

[0509] The proportion of ATRA- cells in the spleen from the mediator group was greater than that of ATRA+ cells (Table 11), while the proportion of ATRA+ cells in the lamina propria (LP) of the ileum was greater than that of ATRA- cells (Table 13), which confirms that ATRA+ cells preferentially home to the intestine, as expected for this group.

[0510] Compared with the mediated mice, the anti-VLA-4 group had approximately 1 / 10 of the ATRA+ cell proportion in LP and approximately 1 / 2 of the ATRA+ cell proportion in PP (Tables 13 and 12), which confirms that this treatment reduced the intestinal homing of ATRA+ cells, as expected for this positive control.

[0511] Compared to the mediator group, the anti-VLA-4 group showed significantly fewer cells isolated from the Peyer's node and ileal lamina propria (Table 9). This is typically observed in mice treated with anti-VLA-4, particularly in the Peyer's node.

[0512] The proportion of ATRA+ cells in the spleen of this group was significantly higher than that in the mediator group. This is typically observed in mice treated with anti-VLA-4 and is likely due to ATRA+ cells being prevented from homing to the gut and thus accumulating in the spleen.

[0513] The proportion of ATRA+ cells in the LPs of mice from the compound A group was found to be lower than that in the LPs of mice from the mulch treatment group. This reduction was dose-dependent and approached statistical significance for cells treated with 1000 nM compound A.

[0514] Compared with the mediator group, the 1000 nM compound A group had significantly fewer cells isolated from the spleen, while the 100 nM and 1000 nM groups had significantly fewer cells isolated from the Peyer's nodes (Table 9).

[0515] Compared with the mediator group, the proportion of CD45.1+ cells in the spleen and LP in the compound A, 1000 nM group was also significantly smaller (Table 10).

[0516] Overall, these results suggest that cells treated with compound A impaired homing to intestinal tissue.

[0517] Example 6

[0518] Compound A inhibits the upregulation of integrin β7.

[0519] The internalizing activity of peptide compound A was assessed using in vitro assays based on flow cytometry. The results showed that compound A specifically induced α4β7 internalization in human primary cells in a time- and dose-dependent manner. Compound A also reduced α4β7 expression, leading to CD4+ oxidative stress. + T memory cells showed reduced adhesion to MAdCAM1; an average reduction of up to 39% was observed in α4β7 expression, resulting in an average reduction of up to 37% in MAdCAM1 adhesion. Furthermore, after an additional 5 days of incubation following the removal of compound A, the expression of compound A returned to control levels.

[0520] method

[0521] Blood samples from human donors were obtained from the Stanford Blood Center (Stanford, California) according to the IRB-approved research protocol. Blood was aspirated into BD Vacutainer heparin sodium collection tubes (BD Biosciences, catalog number #362753). Peripheral blood mononuclear cells (PBMCs) were isolated from the blood using SepMate-50 tubes and LymphoPrep, according to the manufacturer's protocol. CD4 enrichment was performed on the PBMCs after isolation using the EasySep kit (Stem Cell Technology Corporation), according to the manufacturer's protocol. + T memory cells.

[0522] To determine specificity, human PBMCs were incubated at 37°C for 24 hours with 100 nM of compound C (an analog of compound A) and compound D (an inactive triple mutant peptide analog of compound A) in complete culture medium or without peptides. After incubation, aliquots from each reaction were stained to express α4β7.

[0523] To determine time- and dose-dependent effects, purified human CD4 was subjected to treatment at 37°C. + T memory cells were incubated with 10 nM compound A in complete culture medium for a series of different times (0 h, 1 h, 2 h, 4 h, 6 h, 24 h, 28 h, 30 h, and 48 h) or with different concentrations of compound A (0 nM, 0.01 nM, 0.1 nM, 1 nM, and 10 nM) in complete culture medium for 24 h. After incubation, aliquots of cells from each reaction were stained for α4β7 expression.

[0524] To determine the effects on α4β7 expression and MAdCAM1 adhesion, purified human CD4+ was subjected to treatment at 37°C. + T memory cells were incubated together with various concentrations of compound A (0 nM, 0.01 nM, 0.1 nM, 1 nM, and 10 nM) in complete culture medium for 2 hours. After incubation, the cells were thoroughly washed to remove excess peptides. For each reaction, aliquots of cells were stained for α4β7 expression, and individual aliquots were tested for MAdCAM1 adhesion.

[0525] To assess recovery after washing, human PBMCs were incubated with 10 nM compound A in complete medium (MnCl2-free) at 37°C for 24 hours. Aliquots of cells were collected 24 hours before and after peptide addition and stained for α4β7 expression. Cells were then thoroughly washed to remove excess peptide and incubated in fresh complete medium (MnCl2-free) for another seven days. Aliquots were stained for α4β7 expression on days 1, 2, 4, 5, and 7 after peptide washing.

[0526] Following peptide incubation, aliquots of each reaction were stained for α4β7 surface expression in preparation for flow cytometry. Cells were stained at 4°C for 30 min, washed twice in DPBS containing 0.5% BSA (PBS / BSA), incubated at 4°C with streptavidin BV421 (1:1000 dilution) for 30 min, washed twice in PBS / BSA, and then resuspended in PBS / BSA for analysis. Where applicable, a "fluorescence minus one" (FMO) sample was used as a staining control.

[0527] Samples were analyzed by flow cytometry on a BD (Franklin Lake, New Jersey) FACSVerse flow cytometer equipped with the following lasers: 405 nm (purple), 488 nm (blue), 561 nm (yellow-green), and 640 nm (red). CD4+ + T memory cells were identified as CD4. + CD45RA - CD197 + Lymphocytes. CD4 cells were identified based on staining with the vedotin-BV421 complex. + α4β7 expression in T memory cells; staining was analyzed using BD FACSuite software version 1.0.5. Where applicable, values ​​were normalized to peptide-free controls and expressed as percentages to allow assessment of changes in the specified parameters. Data were plotted and analyzed using Prism software (version 7; GraphPad, La Jolla, CA).

[0528] result

[0529] Human PBMCs were incubated with 100 nM compounds B and C, or incubated without peptide, and stained to induce α4β7 expression. Incubation with compound B instead of compound C or the peptide-free control resulted in α4β7 internalization. Figure 14 These data indicate that internalization depends on the binding of the peptide to α4β7.

[0530] Purified human CD4+ T memory cells were incubated with 10 nM compound A for a period of time (0 to 48 hours) or at compound A concentrations (0-10 nM) for 24 hours, and stained to express α4β7. The results showed that compound A induced α4β7 internalization over time (…). Figure 15 ) and concentration ( Figure 16 This is not dependent on PBMC. Similar results were obtained using PBMC (data not shown).

[0531] Purified human CD4 + T memory cells were incubated with various concentrations of compound A, followed by washing to remove excess peptides. Individual aliquots from each reaction were stained for α4β7 expression and MAdCAM1 adhesion was tested, and values ​​were normalized to the corresponding “peptide-free” control for each assay. Data revealed that compound A reduced α4β7 expression (ranging from 5.4% to 24.7% reduction, normalized to peptide-free control) and MAdCAM1 adhesion (ranging from 18.4% to 36.4% reduction, relative to peptide-free control) (respectively). Figure 17 and Figure 18 These effects are strongly correlated, exhibiting an R-squared value of 0.968. Figure 19 When using purified human CD4 from a second donor. + Similar correlations were obtained when T memory cells were repeatedly assayed (R-squared = 0.940, complete data not shown). As shown in Table 1, data from both donors produced a mean maximum reduction of α4β7 expression of 39% and a mean maximum reduction of MAdCAM1 adhesion of 37%.

[0532] Table 14: Maximum reduction in α4β7 expression and adhesion to MAdCAM1

[0533]

[0534] *Percentage normalized to peptide-free control.

[0535] Human PBMCs were incubated in MnCl2-free medium with 10 nM compound A or peptide-free medium for 24 hours, followed by washing to remove excess peptide and resuspending in fresh MnCl2-free medium. At 24 hours, α4β7 expression, as measured by MFI (5090 MFI), showed only a 20.6% difference compared to the FMO control (4219 MFI). After removing compound A, incubation was continued, and aliquots were removed and stained on days 1, 2, 4, 5, and 7 to measure α4β7 expression. The results showed that, in the presence of peptide, the downregulation of α4β7 expression almost recovered to control levels after an additional 4–5 days of incubation. Figure 20 ).

[0536] in conclusion

[0537] This study demonstrates that compound A specifically induces α4β7 internalization in human primary cells in a time- and dose-dependent manner. The reduction in α4β7 expression by compound A is associated with CD4+. + The reduction in MAdCAM1 adhesion in T memory cells was highly correlated. Compound A required an additional 4-5 days of incubation on cells to fully restore α4β7 expression to control levels.

[0538] Example 7

[0539] Randomized, double-blind, placebo-controlled studies of single and multiple escalation doses of compound A in healthy volunteers

[0540] Ulcerative colitis is a chronic inflammatory bowel disease characterized by remission and relapse, and is marked by bloody diarrhea, abdominal cramps, and fatigue. Its pathogenesis is believed to be caused by an inappropriate immune response to gastrointestinal antigens and environmental triggers in genetically susceptible individuals.

[0541] α4β7 integrin, present on the cell surface of circulating memory T and B lymphocytes, is primarily involved in recruiting leukocytes to the gastrointestinal mucosa and associated lymphoid tissues. The major ligand of α4β7, the mucosal addressing cell adhesion molecule (MAdCAM1), is selectively expressed on the endothelium of the gastrointestinal vascular system and is present at increased concentrations in inflamed tissues.

[0542] Vedolizumab is an intravenously administered humanized IgG monoclonal antibody targeting α4β7, approved for the treatment of moderate to severe ulcerative colitis and Crohn's disease in adult patients unresponsive to one or more conventional therapies, such as steroids, immunosuppressants, or tumor necrosis factor (TNF) inhibitors. Due to the inconvenience and potential systemic risks of injectable therapies, oral GI-restricted therapy selectively targeting α4β7 integrin could offer significant benefits to patients with ulcerative colitis. Compound A is an orally stable peptide that specifically binds to α4β7 integrin on leukocytes and has shown minimal systemic absorption (<1%) in animal studies. This study investigated the safety, tolerability, pharmacokinetics, and pharmacodynamics of oral compound A in healthy male subjects.

[0543] Two pharmacokinetic / pharmacodynamic studies were conducted in healthy volunteers. Study 1 was a first-in-human study in which 40 men received compound A, 100 mg to 1400 mg or placebo as a single dose, and 57 men received compound A, 100 mg to 1000 mg or placebo as multiple doses. Study 2 was a randomized crossover study that compared compound A as a liquid solution and as an immediate-release tablet in 10 subjects with multiple doses of 450 mg twice daily.

[0544] No subjects discontinued treatment due to adverse events. Consistent with the gastrointestinal limiting nature of peptides, systemic exposure was minimal; AUC increased approximately dose-proportional. Once-daily dosing resulted in minimal cumulative effects, and no time-dependent changes in pharmacokinetics were observed. Administration of compound A after a high-fat meal reduced peak plasma concentrations and AUC. Minimal urinary excretion of the intact drug was observed (<0.1%), and fecal excretion of the intact compound A increased in a dose-related manner. A dose-dependent increase in blood receptor occupancy and a decrease in blood receptor expression were observed, supporting target engagement. Twice-daily dosing produced sustained receptor occupancy and low plasma variability (143%).

[0545] Compound A is generally well tolerated after single and multiple oral doses, with low systemic exposure. Twice-daily dosing produces sustained pharmacokinetic and pharmacodynamic results, supporting further investigation in efficacy studies.

[0546] method

[0547] Research Design

[0548] Two studies were conducted at a single clinical center.

[0549] Study 1 was the first human three-part study conducted in healthy male volunteers to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of a liquid solution formulation of compound A.

[0550] Part 1 was a randomized, placebo-controlled, double-blind study of compound A with a single escalation dose in 40 men divided into four equal cohorts. Dose escalations started at 100 mg, 300 mg, 1000 mg, and 1400 mg. Subjects in the 300 mg cohort received treatment once on a fasting basis, followed by a second treatment after a high-fat meal. The high-fat meal consisted of two eggs fried in butter, two strips of bacon, two slices of toast with butter, four ounces of hash browns, and 240 ml of whole milk. During Part 1, subjects fasted for 10 hours before and 4 hours after administration, except for subjects in the 300 mg cohort who ate during the treatment period.

[0551] Part 2 was a randomized, placebo-controlled, double-blind, multiple-escalation dose study conducted in 50 male subjects equally divided into five cohorts. Subjects received either compound A or placebo once daily for 14 days. The doses evaluated in Part 2 included 100 mg, 300 mg, and 1000 mg. During Part 2, subjects in two cohorts (100 mg and 300 mg) received food approximately 30 minutes before each dose, while subjects in the other two cohorts (300 mg and 100 mg) fasted for 10 hours before and 1 hour after administration. In a separate cohort of nine subjects in Part 2, a crossover study was conducted to assess the effect of meal timing on the pharmacokinetics and pharmacodynamics of compound A. Subjects in this cohort received food 30, 60, or 90 minutes after administration of compound A.

[0552] Part 3 was an open-label, randomized, crossover multiple-dose comparison of compound A administered as a liquid solution at 900 mg once daily and 450 mg twice daily for five days. Subjects in Part 3 fasted for 10 hours before and 1 hour after administration of compound A.

[0553] The second study was a 5-day, multiple-dose pharmacokinetic and pharmacodynamic study that compared compound A administered twice daily at 450 mg as a liquid formulation and as a tablet formulation in healthy men and women. Subjects fasted for 10 hours before and 1 hour after the morning dose, and for 1 hour before and after the evening dose.

[0554] The study protocols, participant information, and informed consent forms were reviewed and approved by an independent ethics committee for human research. These studies were conducted in accordance with the Declaration of Helsinki on Biomedical Research Involving Human Participants and the International Conference on Harmonization Good Clinical Practice guidelines, and all research procedures were performed by scientifically and medically qualified personnel. Prior to any research-related activity, participants were provided with written informed consent explaining the nature, purpose, and potential risks and benefits of the study.

[0555] Study subjects

[0556] Both studies used similar screening and recruitment procedures. Participants were screened within 21 days of recruitment. Eligible participants were between 18 and 55 years of age and had a body mass index (BMI) of 18-30 kg / m². 2Between [the two groups], the individuals were in good overall health with no significant medical history or clinically significant abnormalities on physical examination. The first human study (Study 1) recruited only men, while the study evaluating the tablet formulation (Study 2) recruited men and women who consented to use the highly effective contraceptive method based on the Clinical Trial Promotion and Coordination Group during the study period and for 90 days after the last dose.

[0557] Subjects were excluded if they had a history of clinically significant endocrine, gastrointestinal, cardiovascular, hematological, hepatic, immunological, renal, respiratory, or genitourinary abnormalities or diseases, or if they had clinically significant laboratory abnormalities, including impaired renal function (serum creatinine >106 μmol / L or estimated creatinine clearance <80 mL / min) or alanine aminotransferase or aspartate aminotransferase values ​​>1.2 times the upper limit of normal.

[0558] program

[0559] Study 1: The single and multiple escalation dose phases of this study consisted of consecutive dose escalations in 10 subjects per dose cohort. Participants were randomly assigned in an 8:2 ratio to receive either compound A as a 60 mL oral solution or a matched placebo. Dosage solutions were prepared weekly by a qualified pharmacist in 50 mM phosphate-buffered saline at pH 7.4. Dosage solutions exceeding the expected concentration range were proven stable for 3 months when stored at 2–8°C.

[0560] Blood samples were collected before administration and 48 hours after a single dose for pharmacokinetic purposes. During multiple dose escalation phases, blood samples were obtained on days 1–3 and 14–16; on day 8, samples were obtained before administration, 4 hours, and 12 hours after administration. On day 10 of the MAD, subjects were asked to collect all urine samples at intervals of 0–6 hours, 6–12 hours, 12–18 hours, and 18–24 hours after administration, and on day 11, subjects were asked to collect stool samples.

[0561] Researchers and the safety oversight committee make the decision to proceed to the next dose level based on acceptable safety and tolerability of the lower dose.

[0562] Study 2: This study was a randomized, open-label, two-treatment, two-time-period, multiple-dose study to determine the safety, tolerability, pharmacokinetics, and pharmacodynamics of immediate-release (IR) tablets and liquid solutions of compound A. This study allowed for comparison of the solid dosage form with the liquid formulation studied in a first-in-human study. Subjects were randomized to receive 450 mg of compound A twice daily (BID) for 5 days, administered every 12 hours, consisting of one 300 mg and one 150 mg dose-strength IR tablet, and also received 450 mg of compound A BID as a liquid solution for 5 days, administered every 12 hours.

[0563] Dosage

[0564] The starting doses for the first single-dose and multiple-dose studies in humans were based on the unobserved level of effect (NOEL) from 28-day toxicology studies in rats and cynomolgus monkeys, taking into account the receptor occupancy observed in cynomolgus monkeys. Using standard allometric growth ratios and a 10-fold safety margin, the NOELs determined in rats and monkeys were converted to a human equivalent dose of approximately 145 mg. A starting dose of 100 mg was chosen, and initial doses were gradually increased by approximately 3-fold.

[0565] The dose selected for Study 2 (comparing tablet and oral solution formulations) was based on the pharmacokinetic and pharmacodynamic characteristics of part 3 of Study 1 and the planned expected dose in the efficacy study in patients with moderate to severe ulcerative colitis.

[0566] Analytical methods

[0567] The concentrations of compound A in plasma, urine, and fecal samples from Study 1, and plasma and urine samples from Study 2, were determined using a validated high-performance liquid chromatography-tandem mass spectrometry (LC / MS / MS) method. The drug and internal standard were extracted from the matrices via a protein precipitation procedure. The limits of quantitation in plasma, urine, and feces were 0.2 ng / mL, 20 ng / mL, and 100 ng / mL, respectively. Sample stability was demonstrated for at least 100 days and 4 freeze-thaw cycles for all matrices. The determination coefficient of the calibration curves was at least 0.99 for all matrices. The inter-assay accuracy (deviation %) for plasma ranged from -2.2% to 1.0%, for urine from -3.8% to 9.0%, and for feces from -5.0% to 5.2%. Inter-analytical precision (CV%) for plasma ranged from 3.7% to 7.7%, for urine from 2.8% to 7.0%, and for stool from 1.2% to 5.2%. Reanalysis of samples indicated that >88% of the samples that underwent valid reanalysis met acceptable criteria, indicating that the analytical method was acceptable.

[0568] Study endpoints

[0569] The primary endpoint, the first-in-human study, was to assess the safety and tolerability of compound A after single and multiple doses. Secondary objectives included characterizing pharmacokinetics and pharmacodynamics, evaluating the effect of a high-fat diet on the pharmacokinetics of compound A, and comparing twice-daily and once-daily dosing. Safety assessments, adverse events, and laboratory evaluations for placebo and each dose of compound A are descriptively summarized.

[0570] The endpoint of the second study comparing oral solutions and tablet formulations was pharmacokinetics and pharmacodynamics.

[0571] Pharmacokinetic analysis

[0572] Pharmacokinetic parameters were estimated using a non-compartmental method with Phoenix WinNonlin (Certara, Princeton, NJ). Peak plasma concentration (C0.05) was determined. max ) and the time to reach peak plasma concentration (T) max The observed values ​​are used. The elimination rate is estimated based on the slope of the least squares regression in the final log-linear stage. The AUC is estimated from time zero to the final quantifiable concentration using the linear trapezoidal method. t The area under the plasma concentration-time curve (AUC) is calculated by dividing the final quantifiable concentration by the elimination rate, and then extrapolating the AUC to infinity. ∞ The fluctuation of steady-state plasma concentration is calculated as follows:

[0573] Pharmacodynamic assay of α4β7 receptor occupancy and receptor expression

[0574] Through its use in preclinical studies and clinical trials of vedolizumab, conversion biomarkers such as receptor occupancy have been validated as pharmacodynamic biomarkers. 27-28In this study, a flow cytometry-based assay was designed to quantify the amount of α4β7 integrin on cell surfaces occupied by compound A, or the amount of α4β7 expressed on the cell surfaces of circulating lymphocytes in response to compound A binding. Briefly, in this assay, each heparinized whole blood sample was first treated with a saturated amount of unlabeled competitive peptide serving as a “blocking” control (100% receptor occupancy), or peptide-free samples serving as “unblocked” samples to measure the blocking level of orally administered compound A. After incubation, the blood was stained with a subsaturated concentration of Alexa647-labeled peptide, followed by staining with a panel of cell surface markers (CD45, CD3, CD4, CD45RA, CD19, IgD, and the anti-α4β7 antibody vedolizumab). After staining, the samples were treated with erythrocyte lysis and fixation buffer, washed, and collected on a flow cytometer. To quantify receptor occupancy on memory CD4 T cells expressing α4β7, the intermediate fluorescence intensity (MFI) of the Alexa647-labeled peptide in vedozizumab + memory CD4+ T cells was used. Receptor occupancy was calculated using the following formula: [RO percentage] = (1 – ([unblocked] – [blocked]) / ([baseline unblocked] – [baseline blocked])) × 100.

[0575] α4β7 expression was limited by the mFI of vedolizumab in memory CD4+ T cells from unblocked samples. Receptor expression (RE) was calculated as the percentage change in MFI from baseline vedolizumab staining.

[0576] Statistical analysis

[0577] No formal sample size estimation was performed. In each dose cohort of the single-dose and multiple-escalation-dose studies, eight subjects received oral compound A and two subjects received placebo. Ten subjects were recruited for a second study comparing immediate-release tablet formulations with oral solutions. Recruitment for each study was deemed sufficient to assess tolerability and safety and to allow for characterization of the pharmacokinetics and pharmacodynamics of compound A.

[0578] result

[0579] Subject characteristics and tendencies

[0580] A total of 97 healthy male participants were recruited for Study 1, with 40 recruited for the single-dose phase and 57 recruited for the multiple-dose phase. 95 participants completed their scheduled administration of either compound A or placebo. Two participants withdrew their consent for personal reasons unrelated to safety; one participant did not wish to remain in the clinical unit, and another experienced discomfort with the intravenous cannula. The mean age in the single-dose phase was 28.7 years, and the mean age in the multiple-dose phase was 30.9 years.

[0581] Ten participants were recruited for Study 2, and nine completed both treatments. One participant discontinued the study after day 1 of oral solution treatment due to an adverse event of acute tonsillitis deemed unrelated to the study drug.

[0582] Safety and tolerability

[0583] During the single-dose escalation phase, 14 subjects reported a total of 23 TEAEs. Of the 13 subjects who experienced TEAEs, 12 received compound A (21 events) and 2 received placebo (2 events). All TEAEs were mild to moderate, except for severe headaches considered treatment-independent in subjects treated with 100 mg of compound A. All subjects recovered from the AEs, and no subjects withdrew due to AEs. No clinically relevant changes were observed in respiratory rate or vital signs, clinical laboratory parameters (hematology, coagulation, serum chemistry, or urinalysis), or in the interpretation of electrocardiograms or QTc intervals.

[0584] Safety and tolerability

[0585] Thirty subjects in the multiple-dose group receiving compound A reported a total of 68 adverse events (AEs). Except for two events, all events were mild in severity. One report of an upper respiratory tract infection was characterized as moderate, and one report of influenza occurring after discharge from a clinical unit was classified as severe and considered a serious adverse event. Four subjects receiving placebo reported a total of six mild treatment-associated adverse events (TEAEs), primarily gastrointestinal symptoms. Treatment-associated adverse events reported by two or more subjects in the multiple-dose escalation phase included abdominal discomfort, flatulence, upper respiratory tract infection, back pain, dizziness, and headache. Neurological symptoms, especially headache, were the most frequently reported TEAEs. No clinically relevant changes were observed in respiratory rate, vital signs, clinical laboratory parameters, or electrocardiogram.

[0586] Safety and tolerability

[0587] Of the 10 participants recruited for Study 2, immediate-release tablets were compared to oral solutions, and nine participants completed both treatments. One participant experienced a treatment-independent moderate tonsillitis adverse event, leading to study discontinuation. The incidence of treatment-related adverse events was similar in both treatments. The most common adverse event was headache, and only one participant reported all other adverse events.

[0588] Safety and tolerability

[0589] Pharmacokinetics

[0590] Figure 21The mean plasma concentration-time curves after a single dose of compound A are presented in Table 15. The single-dose pharmacokinetics of compound A are summarized in Table 15.

[0591] Table 15: Single-dose pharmacokinetics of compound A (mean ± SD)

[0592]

[0593] a Median (Minimum, Maximum)

[0594] b N=4

[0595] c No report due to insufficient data.

[0596] d N=7

[0597] The median time to reach peak plasma concentration was 2 to 4 hours. As the dose of compound A increased from 100 mg to 1400 mg, the median peak plasma concentration of compound A (C0) also increased. max The concentration was increased from 2.11 mg / mL to 23.5 ng / mL, and the AUC... inf The concentration was increased from 16.5 ng·h / mL to 260 ng·h / mL. Within the dose range of 100 mg to 1400 mg of compound A, the AUC... inf It increases proportionally to the dose, and C max The dose-proportioning increase was slightly lower. The mean elimination half-life for the lower doses (100 mg and 300 mg) was 3.1 to 4.0 hours, and the mean elimination half-life for the higher doses (1000 mg and 1400 mg) was 5.3 to 5.7 hours.

[0598] Safety and tolerability

[0599] Table 16 summarizes the pharmacokinetics of compound A after multiple doses.

[0600] Table 16: Pharmacokinetics of Compound A at Multiple Doses (Mean ± SD)

[0601]

[0602] a Median (Minimum, Maximum)

[0603] b N=1

[0604] c No report due to insufficient data.

[0605] d N=7

[0606] C10 in the 100 mg and 300 mg dose groups under the condition of eating on day 14, and between the 300 mg and 1000 mg dose groups under the condition of fasting on day 14. max and AUC inf It generally increases in a dose-proportional manner. The median time to reach peak plasma concentration ranges from 2 to 4 hours. The mean elimination half-life is 5.2 to 7.7 hours. Consistent with the half-life, individual subjects showed improvement at C60 levels at 300 mg and 1000 mg on days 1 and 14. max and AUC t The comparison of values ​​showed that the cumulative effect of once-daily dosing was minimal (≤30%). The comparison of the AUCinf value on day 1 and the AUCt value on day 14 indicated that there was no time-dependent change in the pharmacokinetics of compound A.

[0607] During the multiple dose escalation phases, 24-hour urine and fecal samples were collected in the 300 mg and 1000 mg dose groups. Only a small fraction of compound A was completely recovered in urine within 24 hours, with recoveries of 0.028%, 0.056%, and 0.056% in the 300 mg fasting, 300 mg fed, and 1000 mg dose groups, respectively. In the 300 mg fasting, 300 mg fed, and 1000 mg dose groups, 24-hour fecal recovery of compound A showed a dose-related increase, with complete recovery rates of 0.73%, 1.78%, and 16.8%, respectively.

[0608] The effects of food

[0609] During the single-dose escalation portion of Study 1, the effects of a high-fat meal on the pharmacokinetics of 300 mg of compound A were assessed in a crossover manner.

[0610] Administration of compound A within 30 minutes of consuming a high-fat meal reduced peak concentrations and exposure compared to a fasting state (Table 13). The mean peak plasma concentrations of compound A were 6.55 ng / mL and 1.58 ng / mL in the fasting and eating states, respectively. The median time to reach peak concentrations was delayed by one hour after a high-fat meal.

[0611] In Study 1, the effect of the interval between compound A administration and meal consumption was examined. Subjects received meals 30, 60, or 90 minutes after a single 300 mg dose of compound A. For the 30, 60, and 90-minute treatment groups, the median time to reach peak plasma concentrations of compound A was 1 hour, 2 hours, and 4 hours, respectively. After compound A administration, compared to a 30-minute delay, when food was delayed by 60 or 90 minutes, C... max and AUC tSmall increases were observed in the values, with subtle differences noted between 60-minute and 90-minute delays. Based on the more favorable Cmax and AUCt values ​​noted for a 60-minute food delay compared to a 30-minute delay, dosing of additional cohorts of multiple escalation doses included a one-hour fasting interval before and after compound A administration.

[0612] Table 16 presents a comparison of the pharmacokinetics of 300 mg of compound A after overnight fasting versus fasting within 1 hour of administration, as part of multiple escalation dose phases in Study 1. The median time to peak concentration was 4 hours after overnight fasting, compared to 2 hours when food was consumed 1 hour after administration of compound A. Peak plasma concentrations were lower after overnight fasting when food was given 1 hour after administration compared to when food was given 1 hour after administration (Cmax was 7.23 ng / mL on day 1 fasting and 2.32 ng / mL on day 1 with food given 1 hour after administration).

[0613] Pharmacokinetics of once-daily and twice-daily administration

[0614] In Part 3 of Study 1, the efficacy of the dosing regimens was evaluated in a randomized crossover manner after administration of 900 mg once daily for 5 days and 450 mg twice daily for 5 days.

[0615] Figure 25 Mean plasma concentration-time curves for compound A after administration of 900 mg once daily and 450 mg twice daily are presented. Table 17 presents a summary of the pharmacokinetic profiles comparing once-daily and twice-daily administration.

[0616] Table 17: Pharmacokinetics of Compound A after once-daily and twice-daily dosing (mean ± SD)

[0617]

[0618] a Median (Minimum, Maximum)

[0619] b No report due to insufficient data.

[0620] For both dosing regimens, a median peak concentration of 2 hours was observed on days 1 and 5. The steady-state peak concentration was 14.2 ng / mL for once-daily dosing and 9.96 ng / mL for twice-daily dosing. The area under the curve for dose adjustment during the dosing interval was comparable for both treatment regimens. Consistent with the half-life of compound A, the cumulative effect was minimal with once-daily dosing and approximately 1.6 to 1.7 times with twice-daily dosing. Compared to 900 mg once daily, 450 mg of compound A administered twice daily in liquid solution produced sustained plasma concentrations, reflected in lower peak-to-trough variability (143% vs. 245%) and higher trough concentrations (3.25 ng / mL vs. 1.78 ng / mL) (Table 18).

[0621] Pharmacokinetics of liquid solutions and immediate-release tablet formulations of compound A

[0622] Table 18 summarizes the steady-state pharmacokinetics of immediate-release tablets of compound A administered twice daily for 5 days at 450 mg, compared to the liquid solution used in the first human study.

[0623] Table 18: Steady-state pharmacokinetics of compound A after oral administration of 450 mg twice daily as a liquid solution and as an IR tablet (mean ± SD)

[0624]

[0625] a Median (Minimum, Maximum)

[0626] Figure 23 A presents the mean steady-state plasma concentration-time curves for both formulations. The median time to peak concentration (2 hours) was similar for both formulations, but the peak concentration of the IR tablets was approximately 20% lower than that of the liquid solution. The IR tablet formulation has approximately 85% bioavailability relative to the liquid solution. Twice-daily dosing of the tablet formulation yields C... max Approximately 2 times and 1.6 times the AUC-based cumulative concentration. The steady-state valley concentrations of compound A in IR tablets and liquid solutions were comparable (1.86 ng / mL and 1.98 ng / mL, respectively).

[0627] Pharmacodynamics

[0628] Table 19 summarizes the α4β7 levels measured by average receptor occupancy percentage and average receptor expression after a single dose of compound A. + Memory CD4 + Mean pharmacodynamics of T cells.

[0629] Table 19: Pharmacodynamics after a single dose of compound A (mean ± SD).

[0630]

[0631] RO max For maximum receptor occupancy, RE max For maximum receptor expression

[0632] The mean percentage of receptor occupancy and the time course of mean receptor expression after a single dose are as follows: Figure 22 As shown in AB. Peak α4β7 memory CD4 is reached. + The mean time to T-cell receptor occupancy was approximately 4 hours. The mean peak receptor occupancy increased in a dose-related manner, ranging from 61.8% at 100 mg to 94.8% at 1400 mg. The peak receptor occupancy was similar in the 1000 mg and 1400 mg dose cohorts, indicating that receptor occupancy saturation was achieved with a single dose of approximately 1000 mg of compound A. Receptor expression (RE) max The average change in the concentration of compound A with increasing dose ranged from -28.2% for the 100 mg dose group to -49.0% for the 1400 mg dose group (Table 19).

[0633] Table 20 presents the results of multiple doses of compound A followed by α4β7. + Memory CD4 + T cell receptor occupancy percentage.

[0634] Table 20: Multiple-dose pharmacodynamics of compound A (mean ± SD)

[0635]

[0636]

[0637] RO max For maximum receptor occupancy, RE max For maximum receptor expression

[0638] The mean peak receptor occupancy of memory T cells following multiple doses of compound A peaked at approximately 4 hours. The mean receptor occupancy percentage on day 1 in the multiple-dose cohort was comparable to that in the single-dose cohort. On day 1, the mean peak receptor occupancy after 300 mg and 1000 mg was 77.8% and 91.3%, respectively. With continuous daily administration for more than 14 days, the peak receptor occupancy percentage increased slightly. On day 14, the mean peak receptor occupancy after 300 mg and 1000 mg was 79.7% and 95.6%, respectively.

[0639] Administration of compound A within 30 minutes of consuming a high-fat meal reduced pharmacodynamic effects, consistent with the pharmacokinetic effects of a high-fat meal. Peak receptor occupancy percentage (POP) was 83.4% after 300 mg of compound A in a fasting state, compared to 61.4% when administered within 30 minutes of a high-fat meal. Delaying food intake by 60 minutes after compound A administration improved pharmacodynamic characteristics compared to administration of compound A within 30 minutes of consuming food or after a high-fat meal. The differences in steady-state (day 14) pharmacodynamic effects were relatively small when compound A was administered in a fasting state or when food was given 60 minutes after compound A administration (Table 15).

[0640] As part of the multiple dose escalation phases of Study 1, the pharmacodynamic effects of 900 mg once daily and 450 mg twice daily were examined. Table 21 presents a summary of the pharmacodynamic effects of the dosing regimen on receptor occupancy.

[0641] Table 21: Summary of receptor occupancy pharmacodynamics for once-daily and twice-daily (mean ± SD).

[0642]

[0643]

[0644] a N=7

[0645] On day 1, the mean peak receptor occupancy (AUEC) was 94.5% for the 900 mg once-daily regimen, compared to 86.5% for the 450 mg twice-daily regimen. While both regimens produced similar peak AUEC on day 5 (94.9% for 900 mg QD and 91.9% for 450 mg BID), the twice-daily regimen provided a more sustained pharmacodynamic effect. Notably, the twice-daily regimen had a higher AUEC on day 5 compared to the once-daily regimen. The mean AUEC based on 24-hour action area under the curve (AUEC) on day 5 for the twice-daily and once-daily regimens were 85.3% and 79.2%, respectively. The BID regimen also provided a sustained effect, as indicated by the minimal difference in peak and trough AUEC. Furthermore, on day 5, the inter-individual variability in receptor occupancy at the trough was 11.3%–15.2% with 450 mg BID treatment, compared to 26.3%–33.6% with 900 mg QD treatment, suggesting that the BID regimen has a more consistent effect.

[0646] Figure 23B shows the stable state of compound A after being used as an IR tablet or as a liquid solution twice daily. CD4 + Pharmacodynamics of α4β7 memory T cell receptor occupancy percentage. Table 22 summarizes the pharmacodynamics of receptor occupancy in the steady state.

[0647] Table 22: Steady-state pharmacodynamics of compound A after oral administration of 450 mg twice daily as a liquid solution and as an IR tablet (mean ± SD)

[0648]

[0649] RO max For maximum receptor occupancy, RE max For maximum receptor expression

[0650] Peak receptor occupancy was observed at 4 hours for both formulations. The mean steady-state peak receptor occupancy for the IR tablets was 91.9%, and the mean 24-hour peak receptor occupancy was 83.6%, compared to 93.8% for the liquid solutions and 85.8% for the liquid solutions.

[0651] Pharmacokinetic-Pharmacodynamic Correlation

[0652] The sigmoid Emax (Hill) model was used to characterize the plasma concentration-receptor occupancy relationship of compound A in vivo. Figure 24 Receptor occupancy estimation IC 50 and IC 80 The concentrations were 0.69 ng / mL and 5.9 ng / mL, respectively.

[0653] discuss

[0654] Compound A is an orally administered, gut-restricted peptide that specifically binds to α4β7 integrin on leukocytes. This compound is being developed in a phase 2 study as a potential oral therapy for patients with ulcerative colitis. The peptide's GI-restricted nature and enhanced gastrointestinal stability allow for local action and potentially enhance efficacy while minimizing the likelihood of adverse events associated with systemic exposure.

[0655] The primary objective of these studies was to evaluate the safety / tolerability of compound A after single and multiple administrations. Secondary objectives were to evaluate the pharmacokinetic and pharmacodynamic characteristics of compound A after single and multiple escalating oral doses; to assess the effects of food on pharmacokinetic and pharmacodynamic properties; to compare once-daily and twice-daily administration; and to describe the pharmacokinetic and pharmacodynamic properties of immediate-release formulations of compound A.

[0656] In the first human study, compound A was well tolerated after single doses of up to 1400 mg and multiple doses of up to 1400 mg once daily for 14 days. All treatment-associated adverse events (TEAEs) were mild, except for one reported case of severe headache following a single administration of the lowest dose of compound A (100 mg) and one reported case of influenza following 900 mg once daily. No TEAEs led to subject withdrawal from the study. Treatment-associated adverse events observed in two or more subjects after repeated dosing included abdominal discomfort, flatulence, upper respiratory tract infection, back pain, dizziness, and headache, with headache being the most frequently reported TEAE. Treatment with compound A did not result in any clinically meaningful changes in vital signs or clinical laboratory values, and no evidence of QTc prolongation was observed. The profile of treatment-associated adverse events was not different after twice-daily administration as an IR tablet or as a liquid solution of compound A.

[0657] Following a single oral dose, compound A was moderately absorbed, with peak plasma concentrations observed at approximately 4 hours. The increase in AUC of compound A was approximately dose-proportional, while C... max The increase was slightly less than a dose-proportional increase. Compound A exhibited low systemic exposure after both single and multiple administrations. The terminal half-life was 3.1 to 5.7 hours in the fasting state and 5.2 to 7.7 hours in the fed state. Consistent with the terminal half-life, the accumulation of compound A was approximately 0.9-fold and 1.6-fold when administered once daily and twice daily, respectively. Similar AUC inf AUC on Day 1 and Day 14 t This demonstrates that there is no time-dependent pharmacokinetics (Supplementary Table 4).

[0658] Following compound A administration, α4β7 receptor occupancy increased in a dose-dependent manner, reaching a mean peak occupancy greater than 90% at a dose of 900 mg. Gluttony receptor occupancy was approximately 25.4% and 78.6% after once-daily administration of 100 mg and 1000 mg of compound A, respectively, and 79.2% after twice-daily administration of 450 mg of compound A. These receptor occupancy data indicate that compound A concentrations are maintained at levels sufficient to allow for once- or twice-daily dosing. PK / PD correlations show concentration-dependent receptor occupancy with an asymptote at full receptor occupancy, and IC50 is estimated. 50 The concentration was 0.69 ng / mL, and the IC50 value was... 80 The concentration was 5.9 ng / mL. The estimated IC50 for receptor occupancy to be observed in humans is... 50(0.69 ng / mL) was very advantageously compared with the potency of compound A on memory CD4+ T cells expressing α4β7 isolated from human peripheral blood mononuclear cells into recombinant MAdCAM1 (0.73 ng / mL).

[0659] Systemic concentrations of compound A following oral administration are typically low, consistent with the drug's intestinal restrictive nature and the very low oral bioavailability (<1%) observed in mice and cynomolgus monkeys. Fecal recovery of compound A following oral administration increases in a dose-dependent manner, ranging from approximately 1%–2% at 300 mg to 16.8% at 1000 mg. Compound A is a small disulfide-containing cyclic peptide. Orally administered peptides encounter harsh environments in the gastrointestinal tract, including pH conditions ranging from <2 in the stomach to pH 8 in the duodenum, as well as proteolytic enzymes such as gastric hydrolases (pepsin), pancreatic hydrolases (trypsin, chymotrypsin, elastase, aminopeptidase, and carboxypeptidase A and B), and intestinal brush border membrane-bound enzymes (carboxypeptidase, endopeptidase, and aminopeptidase). 29 The highly acidic environment of the stomach leads to the degradation of peptide drugs by disrupting the stability of their three-dimensional structures. The stability of peptides and proteins in the gastrointestinal tract is an inherent problem associated with oral administration, whether local or systemic. Numerous studies indicate that various factors, such as amino acid sequence, molecular size, and exposure to the gastrointestinal environment (including pH and enzymatic activity), play a crucial role in determining peptide stability and oral absorption potential. Cycloning and N-methylation linked by disulfide bonds provide some resistance to enzymatic degradation and may also improve the oral absorption of compound A.

[0660] The presence of a low, detectable, intact plasma concentration after oral administration indicates that compound A can cross the gastrointestinal wall. Additionally, approximately 0.03% to 0.06% of the drug is completely detectable in urine. Orally administered peptides typically have low oral bioavailability. Although the systemic concentrations of compound A are low, they are sufficient to achieve and maintain a glutathione occupancy greater than 80% after once-daily or twice-daily dosing.

[0661] Administration of compound A within 30 minutes of a high-fat meal reduced its oral absorption. Although there was no directional direct correlation between systemic exposure and fecal reabsorption, data indicated that fecal reabsorption increased in line with the reduced absorption following a high-fat meal.

[0662] The steady-state pharmacokinetic and pharmacodynamic profiles of immediate-release tablet formulations of compound A are generally similar to those used in the liquid formulations used in studies conducted for the first time in humans. A 450 mg dose of compound A administered twice daily as an immediate-release tablet produced sustained pharmacokinetic profiles and an average receptor occupancy of approximately 84%.

[0663] in conclusion

[0664] Compound A was administered to 97 healthy male volunteers. In Part 1, the effects of a single escalation dose of compound A, a maximum daily dose of up to 1400 mg, and food were investigated; in Part 2, multiple escalation doses of up to 1000 mg once daily for up to 14 days were tested. Additionally, a comparison was made between 900 mg of compound A once daily for 5 days and 450 mg of compound A twice daily for 5 days. The study drug was well tolerated; no dose-limiting toxicities were observed. With one exception, all adverse events were mild to moderate in severity. One case of severe influenza-like adverse event, possibly related to the study drug, was reported approximately 36 hours after the subject received compound A. The diagnosis was confirmed by influenza A swab test. The subject recovered calmly.

[0665] The maximum tolerated dose for both single and multiple administrations was the highest dose tested, with a single dose of 1400 mg and multiple administrations of 1000 mg. Minimal plasma exposure was observed for both single and multiple administrations, confirming that the drug is largely GI-restricted. A dose-dependent increase in blood receptor occupancy and a decrease in receptor expression were observed, thus supporting the target engagement and pharmacological activity of compound A in healthy volunteers.

[0666] To support the use of the tablet formulations, a multiple-dose crossover pharmacokinetic and pharmacodynamic study was conducted in 10 healthy subjects following administration of either an oral solution twice daily for 5 consecutive days or an immediate-release tablet (450 mg twice daily for 5 consecutive days). On average, on day 5, the peak plasma concentration and AUC (approximately 15%–18%) of compound A in the IR tablets were slightly lower than those in the solution, a difference considered clinically insignificant. The mean steady-state peak receptor occupancy of both formulations was >90%, and the mean receptor occupancy based on the 24-hour area under the curve (AUEC) was comparable between the two formulations on day 5.

[0667] Compound A was safe and well-tolerated when administered orally to healthy subjects over a wide dose range, following single and multiple escalation doses. Consistent with GI-restricted peptides, compound A has low systemic exposure, and its pharmacokinetic profile supports once- or twice-daily dosing. Twice-daily dosing of compound A produces sustained receptor occupancy. The safety, tolerability, and PK / PD profile of compound A in healthy subjects support continued clinical evaluation of this novel gastrointestinal-restricted targeted therapy for inflammatory bowel disease.

[0668] Example 8

[0669] A randomized, double-blind, placebo-controlled study evaluating the safety and efficacy of oral compound A in subjects with moderate to severe active ulcerative colitis.

[0670] A phase 2 randomized, double-blind, placebo-controlled clinical trial was conducted in human patients with moderate to severe ulcerative colitis to demonstrate the safety, tolerability, and efficacy of treatment with oral compound A. The study also evaluated the pharmacokinetic (PK) and pharmacodynamic (PD) and biomarker responses to treatment with oral compound A.

[0671] Research Design

[0672] This is a two-part study: Part 1 is a randomized, double-blind, placebo-controlled, parallel design 12-week induction therapy period in patients with moderate to severe active UC; and Part 2 is a 40-week extension therapy period, which will include subjects who successfully completed Part 1. Subjects who complete Part 1 at the 12-week visit will be eligible to proceed to Part 2.

[0673] Part 1: Induction Therapy Phase (ITP):

[0674] Part 1 is a 12-week randomized, double-blind, placebo-controlled, parallel-design study in adult subjects with moderate to severe active UC. Eligible subjects will be randomized 1:1:1 to compound A 450 mg twice daily (BID), compound A 150 mg BID, or placebo BID. Subjects must have a biopsy-confirmed diagnosis of UC. To meet inclusion criteria, eligible subjects must have had an inadequate initial response, loss of response, or intolerance to older conventional therapies for UC (i.e., corticosteroids, aminosalicylates, or immunomodulators), or an inadequate initial response, loss of response, or intolerance to newer biologic therapies (i.e., TNFα antagonists or IL-12 / 23 antagonists). Subjects with a history of prior vedolizumab treatment will be excluded. Randomization will be stratified based on prior failure to use TNFα antagonists or IL-12 / 23 antagonists.

[0675] Eligible participants must meet the following inclusion criteria:

[0676] • Male and female participants were aged 18 years (or, if >18, the minimum country-specific age of consent) to 75 years;

[0677] • Participants understood the research procedures and consented to participate in the research by providing written informed consent;

[0678] • Support the diagnosis of UC with appropriate documentation of biopsy results consistent with UC;

[0679] • Suffering from moderate to severe active UC; and

[0680] • It has been shown to have an inadequate response, loss of response, or intolerance to at least one of oral aminosalicylate (5-ASA), corticosteroids, immunomodulators, or biologics (excluding vedolizumab).

[0681] Furthermore, eligible subjects do not meet the following exclusion criteria:

[0682] • Subjects are currently diagnosed with Crohn's disease (CD), indeterminate colitis (IC), microscopic colitis, ischemic colitis, or radiation colitis;

[0683] • History of colonic dysplasia, except for completely removed low-grade dysplasia lesions;

[0684] • A history of active bacterial, viral, fungal, or mycobacterial infection requiring hospitalization or IV antibiotic / anti-infective treatment within 4 weeks of screening or oral antibiotic / anti-infective medication within 2 weeks of screening;

[0685] • Prior treatment with vedolizumab, natalizumab, or any agent targeting α4β7 or β1 integrin during the study period;

[0686] • Positive fecal test for Clostridium difficile;

[0687] • Chronic, relapsing, or severe infections;

[0688] • Known primary or secondary immunodeficiency;

[0689] • Pregnant or breastfeeding women, or those considering pregnancy during the study period or within 30 days of the last dose of the study drug; and

[0690] • History of any major neurological disorder.

[0691] Eligible subjects were randomly assigned in a 1:1:1 ratio to receive compound A 450 mg twice daily (BID), compound A 450 mg 150 mg BID, or placebo BID.

[0692] Part 2: Extended Treatment Period (ETP):

[0693] Participants who complete the 12-week follow-up of Part 1, including components of the Adapted Mayo Score, will be eligible to proceed to Part 2. All Part 1 completers will be eligible to proceed to the extended treatment period in Part 2, at the investigator's discretion. Participants will be blinded and assigned to the appropriate extended treatment group. All participants continuing into Part 2 will receive Compound A.

[0694] Test product, dosage, and method of application:

[0695] Compound A (300 mg and 150 mg) and a matching placebo tablet will be administered orally. Both Compound A and the placebo will have the same appearance.

[0696] Results analysis:

[0697] The primary outcome measure includes the proportion of subjects who achieved clinical remission at week 12 compared to placebo. Clinical remission was assessed using the following adaptive Mayo Criterion (the sum of three sub-scores in the Mayo Criterion):

[0698] • Stool frequency sub-score (SFS)

[0699] • Rectal bleeding score (RBS)

[0700] Endoscopic Score (ESS)

[0701] Secondary outcome measures included individual comparisons between high and low doses of compound A and placebo:

[0702] • The proportion of subjects who experienced endoscopic improvement.

[0703] • The proportion of subjects who achieved endoscopic relief.

[0704] • The proportion of subjects with histological improvement.

[0705] • The proportion of subjects who achieved histological remission.

[0706] • The proportion of subjects with mucosal healing.

[0707] Other outcome measures included the proportion of subjects achieving clinical remission at week 52. Clinical remission was assessed using the following adaptive Mayo Criterion (the sum of three sub-scores in the Mayo Criterion):

[0708] • Stool frequency sub-score (SFS)

[0709] • Rectal bleeding score (RBS)

[0710] • Endoscopic score (ESS).

[0711] Efficacy evaluation

[0712] The efficacy assessment is based at least in part on the Mayo Criterion score. The Mayo Criterion score comprises four components: Stool Frequency Subscale (SFS), Rectal Bleeding Subscale (RBS), Endoscopic Surgery (ESS), and Physician Overall Assessment (PGA). Each score in the individual components ranges from 0 to 3, with higher numbers indicating greater severity.

[0713] The complete Mayo score is the sum of all four sub-scores (SFS, RBS, ESS, and PGA) and ranges from 0 to 12.

[0714] The Adaptive Mayo Criterion (AMC) score is the sum of three sub-scores (SFS, RBS, and ESS). The AMC score ranges from 0 to 9.

[0715] • The Mayo score is partly the sum of three sub-scores (SFS, RBS, and PGA), ranging from 0 to 9.

[0716] • Endoscopic score (ESS) ≤ 1 (modified so that 1 point does not include fragility).

[0717] result

[0718] Treatment with any dose of Compound A is expected to be safe, and treatment with 450 mg BID or 150 mg BID will show statistically significant improvement in full Mayo Criterion, adaptive Mayo Criterion and / or partial Mayo Criterion compared with treatment with placebo, thus demonstrating the efficacy of these doses of Compound A in the treatment of ulcerative colitis.

[0719] All of the aforementioned U.S. patents, U.S. patent applications publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications mentioned in this specification and / or listed in the application data sheet are incorporated herein by reference in their entirety.

[0720] The invention may be practiced in other specific forms without departing from the structure, method, or other essential characteristics broadly described herein and claimed below. The described embodiments are to be considered merely illustrative and not restrictive in all respects. Therefore, the scope of the invention is indicated by the appended claims rather than by the foregoing description. All modifications falling within the meaning and scope of equivalents of the claims are to be covered by the claims. sequence list <110> Protagonist Therapeutics, Inc. Cheng, Xiaoli Liu, David Y. Mattheakis, Larry C. Gupta, Suneel Kumar Modi, Nishit Bachulal <120> Methods for treating inflammatory bowel disease using α4β7 integrin antagonists <130> PRTH-052 / 01WO 321085-2350 <150> US 62 / 959,854 <151> 2020-01-10 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Preparation of peptide antagonists in the laboratory <220> <221> MOD_RES <222> (1)..(1) <223> Xaa is N-methylarginine. <220> <221> MOD_RES <222> (1)..(1) <223> N-methyl-arginine modified with 2-methylbenzoyl <220> <221> MOD_RES <222> (6)..(6) <223> Xaa is penicillamine. <220> <221> MOD_RES <222> (7)..(7) <223> Xaa is (S)-2-amino-3-(4-tert-butyl-phenyl)propionic acid. <220> <221> MOD_RES <222> (8)..(8) <223> Xaa is β-homocysteine <220> <221> MOD_RES <222> (9)..(9) <223> D-type Glu <220> <221> MOD_RES <222> (10)..(10) <223> Type D Lys <400> 1 Xaa Ser Asp Thr Leu Xaa Xaa Xaa Glu Lys 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Preparation of peptide antagonists in the laboratory <220> <221> MOD_RES <222> (1)..(1) <223> Xaa is N-methylarginine. <220> <221> MOD_RES <222> (1)..(1) <223> N-methyl-arginine modified with 2-methylbenzoyl <220> <221> MOD_RES <222> (6)..(6) <223> Xaa is penicillamine. <220> <221> MOD_RES <222> (7)..(7) <223> Xaa is (S)-2-amino-3-(4-tert-butyl-phenyl)propionic acid. <220> <221> MOD_RES <222> (8)..(8) <223> Xaa is β-homocysteine <220> <221> MOD_RES <222> (10)..(10) <223> Type D Lys <400> 2 Xaa Ser Asp Thr Leu Xaa Xaa Xaa Gly Lys 1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Preparation of peptide antagonists in the laboratory <220> <221> MOD_RES <222> (1)..(1) <223> Xaa is N-methylarginine. <220> <221> MOD_RES <222> (1)..(1) <223> N-methyl-arginine modified with 2-methylbenzoyl <220> <221> MOD_RES <222> (6)..(6) <223> Xaa is penicillamine. <220> <221> MOD_RES <222> (7)..(7) <223> Xaa is (S)-2-amino-3-(4-tert-butyl-phenyl)propionic acid. <220> <221> MOD_RES <222> (8)..(8) <223> Xaa is β-homocysteine <220> <221> MOD_RES <222> (10)..(10) <223> Type D Lys <400> 3 Xaa Ser Asp Thr Leu Xaa Xaa Xaa Pro Lys 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Preparation of peptide antagonists in the laboratory <220> <221> MOD_RES <222> (1)..(1) <223> Xaa is N-methylarginine. <220> <221> MOD_RES <222> (1)..(1) <223> N-methyl-arginine modified with 2-methylbenzoyl <220> <221> MOD_RES <222> (6)..(6) <223> Xaa is penicillamine. <220> <221> MOD_RES <222> (7)..(7) <223> Xaa is (S)-2-amino-3-(4-tert-butyl-phenyl)propionic acid. <220> <221> MOD_RES <222> (8)..(8) <223> Xaa is β-homocysteine <220> <221> MOD_RES <222> (9)..(9) <223> D-type Pro <220> <221> MOD_RES <222> (10)..(10) <223> Type D Lys <400> 4 Xaa Ser Asp Thr Leu Xaa Xaa Xaa Pro Lys 1 5 10 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Preparation of peptide antagonists in the laboratory <220> <221> MOD_RES <222> (1)..(1) <223> Xaa is N-methylarginine. <220> <221> MOD_RES <222> (1)..(1) <223> N-methyl-arginine modified with 2-methylbenzoyl <220> <221> MOD_RES <222> (6)..(6) <223> Xaa is penicillamine. <220> <221> MOD_RES <222> (7)..(7) <223> Xaa is (S)-2-amino-3-(4-tert-butyl-phenyl)propionic acid. <220> <221> MOD_RES <222> (8)..(8) <223> Xaa is β-homocysteine <220> <221> MOD_RES <222> (9)..(9) <223> Type D Lys <400> 5 Xaa Ser Asp Thr Leu Xaa Xaa Xaa Lys 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Preparation of peptide antagonists in the laboratory <220> <221> MOD_RES <222> (1)..(1) <223> Xaa is penicillamine. <220> <221> MOD_RES <222> (2)..(2) <223> Xaa is N-methylarginine. <220> <221> MOD_RES <222> (7)..(7) <223> Xaa is penicillamine. <220> <221> MOD_RES <222> (8)..(8) <223> Xaa is (S)-2-amino-3-(4-tert-butyl-phenyl)propionic acid. <220> <221> MOD_RES <222> (9)..(9) <223> Xaa is β-homocysteine <220> <221> MOD_RES <222> (10)..(10) <223> Type D Lys <400> 6 Xaa Xaa Ser Asp Thr Leu Xaa Xaa Xaa Lys 1 5 10 <210> 7 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Preparation of peptide antagonists in the laboratory <220> <221> MOD_RES <222> (1)..(1) <223> Xaa is penicillamine. <220> <221> MOD_RES <222> (2)..(2) <223> Xaa is N-methylarginine. <220> <221> MOD_RES <222> (7)..(7) <223> Xaa is penicillamine. <220> <221> MOD_RES <222> (8)..(8) <223> Xaa is (S)-2-amino-3-(4-tert-butyl-phenyl)propionic acid. <220> <221> MOD_RES <222> (9)..(9) <223> Xaa is β-homocysteine <220> <221> MOD_RES <222> (11)..(11) <223> Type D Lys <400> 7 Xaa Xaa Ser Asp Thr Leu Xaa Xaa Xaa Pro Lys 1 5 10 <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Preparation of peptide antagonists in the laboratory <220> <221> MOD_RES <222> (1)..(1) <223> Xaa is penicillamine. <220> <221> MOD_RES <222> (2)..(2) <223> Xaa is N-methylarginine. <220> <221> MOD_RES <222> (7)..(7) <223> Xaa is penicillamine. <220> <221> MOD_RES <222> (8)..(8) <223> Xaa is (S)-2-amino-3-(4-tert-butyl-phenyl)propionic acid. <220> <221> MOD_RES <222> (9)..(9) <223> Xaa is β-homocysteine <220> <221> MOD_RES <222> (10)..(10) <223> D-type Pro <220> <221> MOD_RES <222> (11)..(11) <223> Type D Lys <400> 8 Xaa Xaa Ser Asp Thr Leu Xaa Xaa Xaa Pro Lys 1 5 10

Claims

1. Use of an α4β7 integrin antagonist in the preparation of a medicament for treating inflammatory bowel disease (IBD) in a subject in need, wherein said treatment comprises oral administration of the α4β7 integrin antagonist to the subject once or twice daily at a dose of 100 mg to 500 mg, wherein said antagonist is a peptide dimer compound comprising two peptides or a pharmaceutically acceptable salt thereof; wherein each of said two peptides comprises or is composed of the following sequence: (2-Benzyl)-(N-Me-R)-Ser-Asp-Thr-Leu-Pen-Phe(4-tBu)-(β-homo-Glu)-(D-Lys)-OH; Each of the two peptides comprises a thioether bond between a 2-benzyl group and a pen group, the two peptides are linked by a linker moiety that binds to a D-Lys amino acid of the two peptides, the linker moiety being a diethylene glycol acid, and the peptide dimer compound having the following chemical structure: 。 2. The use according to claim 1, wherein the peptide dimer compound or a pharmaceutically acceptable salt thereof is present in doses of 100.0 mg, 112.5 mg, 125.0 mg, 137.5 mg, 150.0 mg, 162.5 mg, 175 mg, 187.5 mg, 200.0 mg, 212.5 mg, 225.0 mg, 237.5 mg, 250.0 mg, 262.5 mg, 275 mg, 287.5 mg, 300.0 mg, 312.5 mg, 325.0 mg, 337.5 mg, 350.0 mg, 362.5 mg, 375 mg, 387.5 mg, 400.0 mg, 412.5 mg, 425.0 mg, 437.5 mg, 450.0 mg, 462.5 mg, 475 mg, 487.5 mg, or 500.0 mg. The subject was given a dose of mg.

3. The use according to claim 2, wherein the peptide dimer compound or a pharmaceutically acceptable salt thereof is administered to the subject at a dose of 150 mg.

4. The use according to claim 3, wherein the dose is administered to the subject twice daily.

5. The use according to claim 2, wherein the peptide dimer compound or a pharmaceutically acceptable salt thereof is administered to the subject at a dose of 450 mg.

6. The use according to claim 5, wherein the dose is administered to the subject twice daily.

7. The use according to claim 1, wherein the pharmaceutically acceptable salt of the peptide dimer compound is an acetate.

8. The use according to any one of claims 1 to 7, wherein the administered dose produces unsaturated blood receptor occupancy (RO) when measured at peak blood or serum levels of the antagonist.

9. The use according to claim 8, wherein when measured at peak blood or serum levels of the antagonist, the administered dose produces less than 90% RO, less than 80% RO, less than 70% RO, less than 60% RO, or less than 50% RO.

10. The use according to any one of claims 1 to 7, wherein the treatment inhibits MadCAM1-mediated T cell proliferation in the gastrointestinal tract.

11. The use according to any one of claims 1 to 7, wherein the treatment reduces the cell surface expression of β7 on CD4+ T cells in the gastrointestinal tract.

12. The use according to any one of claims 1 to 7, wherein the treatment: i) Inducing the internalization of α4β7 integrin on CD4+ T memory cells; ii) Reduces the adhesion of MAdCAM1 to CD4+ T memory cells in the gastrointestinal tract; and / or iii) Inhibit T cell homing to the gastrointestinal tract.

13. The use according to claim 12, wherein the treatment inhibits T cell homing to the lamina propria of the ileum, Pierre's nodes, mesenteric lymph nodes, small intestine and / or colon.

14. The use according to any one of claims 1 to 7, wherein the IBD is ulcerative colitis.

15. The use according to any one of claims 1 to 7, wherein the IBD is Crohn's disease.

16. The use according to any one of claims 1 to 7, wherein the treatment produces one or more of the following pharmacokinetic parameters in the plasma of the subject: Cmax is 1-25 ng / mL; Tmax is 1-5 hours; AUC t 10-250 nanograms hour / ml; AUC inf 10-300 nanograms hour / ml; t 1 / 2 It takes 3-10 hours; AUC tau 30-130 nanograms hour / ml; Ctrough concentration is 1-5 ng / mL; The cumulative Cmax is 0.5-2.5 times; and Cumulative AUC t It ranges from 0.5 to 3.0 times.

17. The use according to any one of claims 1 to 7, wherein the treatment produces one or more of the following pharmacodynamic parameters in the plasma of the subject: ROmax is 50-100%; receptor expression changes max -20% to -60%; The average change in receptor expression ranged from -10% to -55%. The steady-state ROmax is 80-100%; Average RO 0-24小时 It ranges from 50% to 95%; Average RO 0-12小时 80-95%; and Average RO 12-24小时 It is 70-90%.

Images