Methods of treating inflammatory bowel disease

Abstract

The present invention relates to pharmaceutical methods, compositions and combinations for the treatment and / or prevention of inflammatory bowel disease (IBD). In particular, the present invention relates to methods and compositions for the treatment of IBD comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to PCT / CN2020 / 113466, filed on September 4, 2020, the contents of which are incorporated herein for all purposes. Technical Field

[0003] This invention relates to methods for treating inflammatory bowel disease (IBD), as well as compositions and pharmaceuticals that can be used to treat IBD. The methods and compositions are based on data showing that renin inhibitors of formula (I) can alleviate the symptoms and manifestations of IBD. Background Technology

[0004] Inflammatory bowel disease (IBD) is typically a chronic condition that significantly impacts quality of life. IBD includes Crohn's disease (CD) and ulcerative colitis (UC). While their exact nature is not fully understood, they are generally believed to involve excessive or abnormal activation of the mucosal immune system. Current treatments for IBD include anti-inflammatory corticosteroids, aminosalicylates (e.g., mesalazine, balsalazine, oxalazine), and immune pathway inhibitors (azathioprine, mercaptopurine, cyclosporine, methotrexate, TNF-α inhibitors), among others. However, some patients do not respond to available treatments, while others initially respond to known regimens but then lose efficacy. Therefore, new treatment approaches for IBD remain needed.

[0005] Recent reports indicate that activation of the renin-angiotensin system (RAS) promotes colitis. Y. Shi, et al., Scientific Reports (Nature) 6, 27552; doi:10.1038 / srep27552 (2016). Following intrarectal infusion of 2,4,6-trinitrobenzenesulfonic acid (TNBS), RenTgMK mice with hepatic overexpression of active renin developed more severe colitis than wild-type controls. Over 50% of the RenTgMK mice died, while all wild-type mice recovered. Compared to wild-type controls, RenTgMK mice also exhibited stronger mucosal TH17 and TH1 / TH17 responses and more severe colonic epithelial cell apoptosis.

[0006] Treatment of these RenTgMK mice with aliskiren (a renin inhibitor administered via intraperitoneal injection) improved this induced colitis in the mice, while treatment with hydralazine (a smooth muscle relaxant similar to aliskiren that lowers blood pressure) did not affect colitis, indicating that the remission of colitis with aliskiren treatment was not related to the common hypotensive effects of aliskiren and hydralazine.

[0007] Aliskiren was the first direct renin inhibitor approved for the treatment of hypertension. While it has been widely used for this purpose, it poses some risks to patients with diabetes and renal insufficiency due to potential nephrotoxicity. It also has a relatively low bioavailability of only 2.5% (…). (Aligellen) tag), and because there are four chiral centers along the extended linear backbone, the synthesis is complex and expensive.

[0008] Aligilen:

[0009] The authors of the Shi study acknowledge that their model system may not be suitable for normal metabolic conditions because the transgenic experimental animals used tend to amplify the effects of RAS inhibitors. They note that these findings may not mean that endogenous RAS plays a role in the development of colitis under “normal conditions.” “The RenTg mouse model is essentially an ‘artificial’ system that amplifies the effects of RAS for research purposes. Whether endogenous RAS plays a role in the development of colitis under normal conditions needs to be addressed… Therefore, our conclusions regarding the inflammatory role of RAS in colitis need to be carefully generalized.” Shi, pp. 7-8. Summary of the Invention

[0010] This invention provides novel treatment methods and compositions for IBD using a direct renin inhibitor of formula (I). This compound exhibits superior bioavailability compared to aliskiren and is more effective as a renin inhibitor. Data presented herein demonstrate that the compound of formula (I) is effective in treating IBD using model systems of “normal” rats and mice (genetically not predisposed to particular sensitivity to RAS activity). Furthermore, the compound of formula (I) has been shown to reduce the release of inflammatory cytokines in colonic tissue from human patients with ulcerative colitis. Additionally, rat data demonstrate the effectiveness of the compound of formula (I) in treating IBD when administered orally.

[0011]

[0012] Compound (I) is a renin inhibitor, but it is unclear whether its effect on IBD is due to renin inhibition, as data in this paper suggest it inhibits the release of several key pro-inflammatory cytokines, including IL-6; the mechanism of action of IBD has not been explored and is likely multifaceted. However, the methods of this invention are believed to work through a mechanism different from currently approved IBD treatments, and therefore they can be used where current therapies have failed, or they can be combined with current IBD therapies to provide new and more effective treatments for patients with IBD.

[0013] On the one hand, this disclosure provides a method for treating inflammatory bowel disease using a compound of formula (I). Unbound by theory, this compound has been shown to be a potent direct inhibitor of renin, reducing levels of pro-inflammatory cytokines, which may contribute to its effectiveness in treating IBD. It possesses pharmacokinetic properties suitable for therapeutic use via oral administration and has now been shown to be effective for the in vivo treatment of inflammatory bowel disease.

[0014] Unbound by theory, compounds of formula (I) are thought to treat IBD through novel mechanisms of action or in combination with mechanisms that complement current therapies. They can be used in conjunction with current IBD therapies or as an alternative for patients who have problems with current IBD therapies or who do not respond adequately to them. IBDs that can be treated with these methods include Crohn's disease and ulcerative colitis. This approach can be used to treat subjects diagnosed with IBD, such as ulcerative colitis or Crohn's disease.

[0015] In some embodiments, the compound of formula (I) is typically administered orally in solid dosage forms such as tablets or capsules. Other suitable formulations include soft capsules for oral administration and suppositories for direct introduction into the colon. Administration may be a single dose or multiple doses, and the dosage of the compound of formula (I) may be administered at least once daily, typically in the form of one, two, or three tablets or capsules, or may be administered every other day, or at least once weekly. In some embodiments, a single dose is administered at least once daily to a subject requiring treatment for ulcerative colitis or Crohn's disease. In other embodiments, a single dose is administered to the subject twice or three times daily. In a preferred embodiment, administration is typically by oral means, with one dose administered twice daily.

[0016] On the other hand, the present invention provides a method as described above, wherein a compound of formula (I) is administered to a subject who is also receiving another IBD therapy, which may be selected, for example, anti-inflammatory corticosteroids, aminosalicylates, and other IBD therapies, including but not limited to:

[0017] a) Anti-TNFα agents (e.g., infliximab, adalimumab, pecelizumab, golimumab);

[0018] b) Sphingosine-1-phosphate (S1P) receptor modulators (e.g., ozamod);

[0019] c) Anti-adhesion (anti-integrin) agents (e.g., nastatinumab, vedotinumab, ertrlizumab);

[0020] d) IL-12 / IL-23 inhibitors (e.g., ustekinumab, risankizumab);

[0021] e) Transforming growth factor β (TGFβ) inhibitors (e.g., Mongersen, Pirfenidone);

[0022] f) Phosphodiesterase 4 (PDE4) inhibitors (e.g., aprestin);

[0023] g) Janus kinase (JAK) / STAT inhibitors (e.g., tofacitinib, filagrinib);

[0024] h) Stem cell transplantation (e.g., hematopoietic stem cells, adipose-derived stem cells);

[0025] i) Fecal microbiota transplantation (FMT);

[0026] j) Plasminogen activator inhibitor-1 (PAI-1) inhibitors (e.g., MDI-2268, titraxtinin);

[0027] k) Aminosalicylates (e.g., mesalazine, balsalazine, olsalazine);

[0028] l) Anti-inflammatory corticosteroids; and,

[0029] m) Immune pathway inhibitors, such as azathioprine, mercaptopurine, cyclosporine, and methotrexate.

[0030] On the other hand, the present invention provides solid dosage forms comprising a compound of formula (I) that can be formulated for the treatment of IBD. The solid dosage forms typically contain 25 mg to 800 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof in a single unit dose formulated for oral administration. In some such embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is formulated as, for example, tablets, capsules, soft capsules, or suppositories, further comprising at least one additional IBD therapeutic agent selected from anti-inflammatory corticosteroids, aminosalicylates, or other IBD therapies, such as:

[0031] Anti-TNFα agents;

[0032] Sphingosine-1-phosphate (S1P) receptor modulators (e.g., ozamod);

[0033] Anti-adhesion (anti-integrin) agents;

[0034] IL-12 / IL-23 inhibitors;

[0035] Transforming growth factor β (TGFβ) inhibitors (e.g., Mongersen, Pirfenidone);

[0036] Phosphodiesterase 4 (PDE4) inhibitors (e.g., aprescribing);

[0037] Janus kinase (JAK) / STAT inhibitors (e.g., tofacitinib, filagrinib); and,

[0038] Plasminogen activator inhibitor-1 (PAI-1) inhibitors (e.g., MDI-2268, tiplaxtinin).

[0039] On the other hand, this disclosure provides a delayed-release formulation of a compound of formula (I) or a pharmaceutically acceptable salt thereof for oral administration. Typically, the delayed-release formulation is configured or designed to pass through the stomach and into the intestine before releasing most or substantially all of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the intestine of a subject, particularly in the colon. The invention also provides a method of treating IBD by administering such a delayed-release formulation to a subject requiring treatment for IBD.

[0040] On the other hand, this disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for treating inflammatory bowel disease. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof is formulated for oral administration to a subject requiring treatment for inflammatory bowel disease. In some such embodiments, the compound or a pharmaceutically acceptable salt thereof is formulated as a delayed-release formulation, designed to pass through the recipient's stomach before most or substantially all of the compound of formula (I) or a pharmaceutically acceptable salt thereof is released into the recipient's intestine. In some such embodiments, most of the compound of formula (I) or a pharmaceutically acceptable salt thereof is released in the colon of the treated subject.

[0041] On the other hand, the present invention provides a method for preparing a medicament for treating inflammatory bowel disease using a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some such embodiments, the medicament is formulated for oral delivery. In some such embodiments, the medicament is formulated as a delayed-release formulation that passes through the stomach of the subject before most or substantially all of the compound of formula (I) or a pharmaceutically acceptable salt thereof is released in the intestine of the subject.

[0042] In another aspect, this disclosure provides combinations for treating and / or preventing inflammatory bowel disease, comprising administering a compound of formula (I) or a pharmaceutically acceptable salt thereof, in addition to treating the subject with at least one other IBD therapy selected from:

[0043] a) Anti-TNFα agents (e.g., infliximab, adalimumab, pecelizumab, golimumab);

[0044] b) Sphingosine-1-phosphate (S1P) receptor modulators (e.g., ozamod);

[0045] c) Anti-adhesion (anti-integrin) agents (e.g., nastatinumab, vedotinumab, ertrlizumab);

[0046] d) IL-12 / IL-23 inhibitors (e.g., ustekinumab, risankizumab);

[0047] e) Transforming growth factor β (TGFβ) inhibitors (e.g., Mongersen, Pirfenidone);

[0048] f) Phosphodiesterase 4 (PDE4) inhibitors (e.g., aprestin);

[0049] g) Janus kinase (JAK) / STAT signal transduction and transcription activator inhibitors (e.g., tofacitinib, filagrinib);

[0050] h) Stem cell transplantation (e.g., hematopoietic stem cells, adipose-derived stem cells);

[0051] i) Fecal microbiota transplantation (FMT);

[0052] j) Plasminogen activator inhibitor-1 (PAI-1) inhibitors (e.g., MDI-2268, titraxtinin);

[0053] k) Aminosalicylates (e.g., mesalazine, balsalazine, olsalazine);

[0054] l) Anti-inflammatory corticosteroids; and,

[0055] m) Immune pathway inhibitors, such as azathioprine, mercaptopurine, cyclosporine, and methotrexate. Attached Figure Description

[0056] Figure 1 The weight of the experimental animals (Wistar rats in a DNBS-induced colitis model) in Example 1 is shown.

[0057] Figure 2 The stool consistency score during the described 7-day test is shown, and the area under the curve (AUC) for each group is used as the effect indicator.

[0058] Figure 3The macroscopic evaluation of the colon at the end of 7 days of treatment in Example 1 is shown, including colon weight (CW), colon length (CL), and ulcer area.

[0059] Figure 4A and 4B The study showed gross morphological differences in the colons of C57BL / 6 mice treated with TNBS to induce colitis, and showed that treatment with compound (I) of formula (“SPH-X”) twice daily at 20 mg / kg malate form after exposure to trinitrobenzenesulfonic acid (TNBS) significantly reversed the damage caused by TNBS.

[0060] Figure 5A and 5B Microscopic evidence of colonic mucosal tissue damage from an induced colitis model was shown, and treatment or prevention of such damage was demonstrated by intraperitoneal administration of SPH-X (a compound of formula (I) in the form of malate) twice daily after exposure to TNBS.

[0061] Figure 6 Exposure to TNBS showed that SPH-X (compound of formula (I) in the form of malate) significantly reduced the overproduction of cytokines IL-1β and IL-6 in colonic mucosal tissue.

[0062] Figure 7 The images are Western blots showing that treatment of colon tissue with TNBS led to an increase in TNF-α levels, and treatment with SPH-X (10 mg / kg twice daily) reduced or stopped TNF-α formation.

[0063] Figure 8A -C shows the effect of Birb 796 and CFN001 / 01 (which identified a specific batch of SPH-X, a compound of formula (I) in the form of malate) on the release of IL-6 from human colon tissue samples from patients with ulcerative colitis (UC): Figure 8A Display data for donor A. Figure 8B Displaying data for donor B, Figure 8C Display the data for donor C. Detailed Implementation

[0064] General definition:

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents, applications, published applications, and other publications mentioned herein are incorporated herein by reference in their entirety. If any definition set forth in this section contradicts or is inconsistent with a definition set forth in a patent, application, or other publication incorporated herein by reference, the definition set forth in this section shall prevail over the definition incorporated herein by reference.

[0066] As used in this article, "a" or "an" means "at least one" or "one or more".

[0067] The term "pharmaceutically acceptable salt" refers to a salt (a salt with an anti-ion that has acceptable mammalian safety for a given dosage regimen) administered to a patient, such as a mammal, like a human. Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. "Pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of a compound derived from a variety of organic and inorganic anti-ions well known in the art, and by way of example only, includes sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc.; and when the molecule contains a basic functional group, salts of organic or inorganic acids, such as hydrochlorides, hydrobromic acids, formates, tartrates, malates, benzenesulfonates, methanesulfonates, acetates, maleates, oxalates, etc.

[0068] The term "its salt" refers to a compound formed when the proton of an acid is replaced by a cation, such as a metal cation or an organic cation. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not essential for salts of intermediate compounds not intended for administration to patients. For example, salts of the compounds of the present invention include salts in which the compound is protonated by an inorganic or organic acid to form a cation, wherein the conjugate base of the inorganic or organic acid serves as the anionic component of the salt.

[0069] Compound of formula (I)

[0070] The structure of compound (I) is shown below. This compound exhibits potent activity as a renin inhibitor and pharmacokinetic characteristics suitable for oral administration. Bioavailability in rats is approximately 11.5–24.5%, and in monkeys, it is approximately 3.3–11.3%. The plasma renin activity of compound (I) is 0.28 nM, while that of aliskiren is 0.60 nM, and the activity is maintained for 24 hours even at a low dose of 0.2 mg / kg.

[0071]

[0072] It can be formulated and administered as a neutral compound or a pharmaceutically acceptable salt. For the experiments described herein, the compound of formula (I) is administered in its malate form. The synthesis and characterization of this compound are disclosed, for example, in U.S. Patent No. 9,278,944. The preparation of the malate is described in U.S. Patent No. 10,519,150. In the methods, compositions, and combinations disclosed herein, the malate of the compound of formula (I) is preferred.

[0073] malate of formula (I)

[0074] On the other hand, this disclosure provides a compound of formula (I) or its malate for treating inflammatory bowel disease.

[0075] In another aspect, the present invention provides a method for preparing a medicament for treating inflammatory bowel disease using a compound of formula (I) or its malate.

[0076] Some aspects of the present invention are summarized in the following list of embodiments.

[0077] 1. A method of treating inflammatory bowel disease in a subject requiring treatment for inflammatory bowel disease, comprising administering to the subject an effective amount of a compound of formula (I).

[0078]

[0079] Or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the compound of formula (I) is used in its malate form.

[0080] 2. The method described in Implementation Scheme 1, wherein the inflammatory bowel disease is ulcerative colitis.

[0081] 3. The method described in Implementation Scheme 1, wherein the inflammatory bowel disease is Crohn's disease.

[0082] 4. The method according to any one of embodiments 1-3, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered orally. In some such embodiments, the compound is administered in the form of tablets, capsules, or soft capsules.

[0083] 5. The method according to any one of embodiments 1-3, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered as a suppository.

[0084] 6. The method according to any one of embodiments 1-5, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to the subject at least once daily. In a preferred aspect of this embodiment, the compound of formula (I) is administered twice daily.

[0085] 7. The method of implementation plan 6, wherein at least one dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered to the subject twice daily.

[0086] 8. The method of any one of embodiments 1-7, wherein the dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof administered to the subject is from 25 mg to 800 mg. In specific examples of this embodiment, the dose is about 25 mg, or 50 mg, or 75 mg, or 100 mg, or 125 mg, or 150 mg, or 175 mg, or 200 mg, or 225 mg, or 250 mg, or 275 mg, or 300 mg, or 350 mg, or 400 mg, or 450 mg, or 500 mg, or 550 mg, or 600 mg, or 650 mg, or 700 mg, or 750 mg, or 800 mg.

[0087] 9. The method according to any one of embodiments 1-8, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered as a delayed-release formulation, preferably a formulation configured to promote the release of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the lower gastrointestinal tract, or a formulation configured to reduce the release of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the stomach.

[0088] 10. The method according to any one of embodiments 1-9, wherein the subject is further treated with at least one additional IBD therapy. The additional IBD therapy may be administered in combination with or separately from the compound of formula (I), and may be administered via the same or different routes of administration.

[0089] 11. The method of embodiment 10, wherein the at least one additional IBD therapy is selected from:

[0090] a) Anti-TNFα agents (e.g., infliximab, adalimumab, pecelizumab, golimumab);

[0091] b) Sphingosine-1-phosphate (S1P) receptor modulators (e.g., ozamod);

[0092] c) Anti-adhesion (anti-integrin) agents (e.g., nastatinumab, vedotinumab, ertrlizumab);

[0093] d) IL-12 / IL-23 inhibitors (e.g., ustekinumab, risankizumab);

[0094] e) Transforming growth factor β (TGFβ) inhibitors (e.g., Mongersen, Pirfenidone);

[0095] f) Phosphodiesterase 4 (PDE4) inhibitors (e.g., aprestin);

[0096] g) Janus kinase (JAK) / STAT signal transduction and transcription activator inhibitors (e.g., tofacitinib, filagrinib);

[0097] h) Stem cell transplantation (e.g., hematopoietic stem cells, adipose-derived stem cells);

[0098] i) Fecal microbiota transplantation (FMT);

[0099] j) Plasminogen activator inhibitor-1 (PAI-1) inhibitors (e.g., MDI-2268, titraxtinin);

[0100] k) Aminosalicylates (e.g., mesalazine, balsalazine, olsalazine);

[0101] l) Anti-inflammatory corticosteroids; and,

[0102] m) Immune pathway inhibitors, such as azathioprine, mercaptopurine, cyclosporine, and methotrexate.

[0103] 12. Compound of formula (I) used to treat inflammatory bowel disease

[0104]

[0105] Or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the compound of formula (I) is used in its malate form.

[0106] 13. The compound of formula (I) or a pharmaceutically acceptable salt thereof for treating inflammatory bowel disease according to embodiment 12, wherein the inflammatory bowel disease is ulcerative colitis.

[0107] 14. The compound of formula (I) or a pharmaceutically acceptable salt thereof for treating inflammatory bowel disease according to embodiment 12, wherein the inflammatory bowel disease is Crohn's disease.

[0108] 15. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of embodiments 12-14, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is prepared for oral administration.

[0109] 16. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of embodiments 12-15, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is prepared to be administered to the subject at least once a week, usually at least once a day, preferably twice a day.

[0110] 17. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to embodiment 16, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is prepared to be administered to the subject at least once daily, preferably twice daily.

[0111] 18. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of embodiments 12-17, wherein the dosage of the compound of formula (I) or a pharmaceutically acceptable salt thereof prepared for administration comprises 25 mg to 800 mg of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In a specific example of this embodiment, the dosage is about 25 mg, or 50 mg, or 75 mg, or 100 mg, or 125 mg, or 150 mg, or 175 mg, or 200 mg, or 225 mg, or 250 mg, or 275 mg, or 300 mg, or 350 mg, or 400 mg, or 450 mg, or 500 mg, or 550 mg, or 600 mg, or 650 mg, or 700 mg, or 750 mg, or 800 mg.

[0112] 19. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of embodiments 12-18, wherein the compound is prepared as a delayed-release formulation.

[0113] 20. The compound of formula (I) according to embodiment 19 or a pharmaceutically acceptable salt thereof, wherein the delayed-release formulation is configured to promote the release of the compound in the lower gastrointestinal tract or to reduce the release of the compound in the stomach.

[0114] 21. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of embodiments 12-20, wherein the compound is prepared or formulated for use in combination with another IBD therapy.

[0115] 22. The compound of formula (I) according to embodiment 21 or a pharmaceutically acceptable salt thereof, wherein the at least one additional IBD therapy is selected from:

[0116] a) Anti-TNFα agents (e.g., infliximab, adalimumab, pecelizumab, golimumab);

[0117] b) Sphingosine-1-phosphate (S1P) receptor modulators (e.g., ozamod);

[0118] c) Anti-adhesion (anti-integrin) agents (e.g., nastatinumab, vedotinumab, ertrlizumab);

[0119] d) IL-12 / IL-23 inhibitors (e.g., ustekinumab, risankizumab);

[0120] e) Transforming growth factor β (TGFβ) inhibitors (e.g., Mongersen, Pirfenidone);

[0121] f) Phosphodiesterase 4 (PDE4) inhibitors (e.g., aprestin);

[0122] g) Janus kinase (JAK) / STAT signal transduction and transcription activator inhibitors (e.g., tofacitinib, filagrinib);

[0123] h) Stem cell transplantation (e.g., hematopoietic stem cells, adipose-derived stem cells);

[0124] i) Fecal microbiota transplantation (FMT);

[0125] j) Plasminogen activator inhibitor-1 (PAI-1) inhibitors (e.g., MDI-2268, titraxtinin);

[0126] k) Aminosalicylates (e.g., mesalazine, balsalazine, olsalazine);

[0127] l) Anti-inflammatory corticosteroids; and,

[0128] m) Immune pathway inhibitors, such as azathioprine, mercaptopurine, cyclosporine, and methotrexate.

[0129] 23. Compound of formula (I)

[0130]

[0131] The use of its pharmaceutically acceptable salts in the preparation of medicaments for the treatment of inflammatory bowel disease.

[0132] In a preferred embodiment, the compound of formula (I) is used in its malate form.

[0133] 24. The use described in Implementation Scheme 23, wherein the inflammatory bowel disease is ulcerative colitis.

[0134] 25. The use described in Implementation Scheme 23, wherein the inflammatory bowel disease is Crohn's disease.

[0135] 26. The use according to any one of embodiments 23-25, wherein the drug is prepared for oral administration or as a suppository.

[0136] 27. The use according to any one of embodiments 23-26, wherein the drug is prepared as a dosage unit, such as a pill, capsule, tablet or soft capsule containing 25 mg to 800 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0137] 28. Use according to any one of embodiments 23-27, wherein the drug is prepared as a delayed-release formulation, preferably a formulation that promotes the release of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the lower gastrointestinal tract or reduces its release in the stomach.

[0138] 29. The use according to any one of embodiments 23-28, wherein the medicament is prepared or formulated for use with at least one additional IBD therapy.

[0139] 30. The use described in embodiment 29, wherein the at least one additional IBD therapy is selected from:

[0140] a) Anti-TNFα agents (e.g., infliximab, adalimumab, pecelizumab, golimumab);

[0141] b) Sphingosine-1-phosphate (S1P) receptor modulators (e.g., ozamod);

[0142] c) Anti-adhesion (anti-integrin) agents (e.g., nastatinumab, vedotinumab, ertrlizumab);

[0143] d) IL-12 / IL-23 inhibitors (e.g., ustekinumab, risankizumab);

[0144] e) Transforming growth factor β (TGFβ) inhibitors (e.g., Mongersen, Pirfenidone);

[0145] f) Phosphodiesterase 4 (PDE4) inhibitors (e.g., aprestin);

[0146] g) Janus kinase (JAK) / STAT signal transduction and transcription activator inhibitors (e.g., tofacitinib, filagrinib);

[0147] h) Stem cell transplantation (e.g., hematopoietic stem cells, adipose-derived stem cells);

[0148] i) Fecal microbiota transplantation (FMT);

[0149] j) Plasminogen activator inhibitor-1 (PAI-1) inhibitors (e.g., MDI-2268, titraxtinin);

[0150] k) Aminosalicylates (e.g., mesalazine, balsalazine, olsalazine);

[0151] l) Anti-inflammatory corticosteroids; and,

[0152] m) Immune pathway inhibitors, such as azathioprine, mercaptopurine, cyclosporine, and methotrexate.

[0153] 31. A pharmaceutical composition comprising a compound of formula (I) mixed with another IBD therapeutic agent.

[0154]

[0155] Or a pharmaceutically acceptable salt thereof. In a preferred embodiment, the compound of formula (I) is used in its malate form.

[0156] 32. The pharmaceutical composition of embodiment 31 is a solid dosage form, soft capsule or suppository for oral administration.

[0157] 33. The pharmaceutical composition according to embodiment 31 or 32, comprising 25 mg to 800 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0158] 34. The pharmaceutical composition according to any one of embodiments 31-33, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is prepared as a delayed-release formulation.

[0159] 35. The pharmaceutical composition according to any one of embodiments 31-34, wherein the pharmaceutical composition is configured to promote the release of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the lower gastrointestinal tract, or is configured to reduce the release of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the stomach.

[0160] 36. The pharmaceutical composition according to any one of embodiments 31-35, wherein the at least one additional IBD therapeutic agent is selected from:

[0161] a) Sphingosine-1-phosphate (S1P) receptor modulators (e.g., ozamod);

[0162] b) Transforming growth factor β (TGFβ) inhibitors (e.g., Mongersen, Pirfenidone);

[0163] c) Phosphodiesterase 4 (PDE4) inhibitors (e.g., aprestin);

[0164] d) Janus kinase (JAK) / signal transduction and transcription activator (STAT) inhibitors (e.g., tofacitinib, fioglotinib);

[0165] e) Plasminogen activator inhibitor-1 (PAI-1) inhibitors (e.g., MDI-2268, tiplaxtinin);

[0166] f) Aminosalicylates (e.g., mesalazine, balsalazine, olsalazine);

[0167] g) Anti-inflammatory corticosteroids; and,

[0168] h) Immune pathway inhibitors (e.g., azathioprine, mercaptopurine, cyclosporine, methotrexate, TNF-α inhibitors).

[0169] In any of the foregoing embodiments, the compound of formula (I) may be used or administered as a malate.

[0170] Pharmaceutical compositions, combinations and other related uses

[0171] In another aspect, this disclosure provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof mixed with at least one pharmaceutically acceptable carrier or excipient, wherein the composition is configured for treating IBD. In some embodiments, the composition further comprises an additional therapeutic agent that can be used to treat IBD. In some embodiments, the pharmaceutical composition is adapted to delay the release of the compound of formula (I) or a pharmaceutically acceptable salt thereof, particularly to promote the release of the compound of formula (I) or a pharmaceutically acceptable salt thereof primarily in the lower gastrointestinal tract and / or reduce the release of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the stomach.

[0172] In another aspect, this disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for the treatment of inflammatory bowel disease. The compound may be used in its malate form.

[0173] In another aspect, this disclosure provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating inflammatory bowel disease. In some of these embodiments, an malate of a compound of formula (I) is used.

[0174] preparation

[0175] Any suitable formulation may be prepared containing a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a combination thereof. See generally Remington's Pharmaceutical Sciences, (2000) Hoover, JE editor, 20th edition, Lippincott Williams and Wilkins Publishing Company, Easton, Pa., pages 780-857. Choose a formulation suitable for the appropriate route of administration. If the basicity or acidity of the compound is sufficient to form a stable, non-toxic acid or basic salt, administration of the compound in salt form may be appropriate. Examples of pharmaceutically acceptable salts are addition salts of organic acids that form physiologically acceptable anions, such as toluenesulfonates, methanesulfonates, acetates, citrates, malonates, tartrates, succinates, benzoates, ascorbic acid salts, α-ketoglutarate, and α-glycerophosphates. Suitable inorganic salts may also be formed, including hydrochlorides, sulfates, nitrates, bicarbonates, and carbonates. Pharmaceutically acceptable salts are obtained using standard procedures known in the art, for example, by using a sufficiently basic compound such as an amine with a suitable acid to provide physiologically acceptable anions. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids are also prepared.

[0176] Preferably, the compound of formula (I) or a pharmaceutically acceptable salt thereof is formulated for oral administration, typically in tablet or capsule form. In some embodiments, an malate of the compound of formula (I) is used.

[0177] When the intended compound is to be administered in the form of a pharmacological composition, it may be considered to formulate the compound by mixing it with a pharmaceutically acceptable excipient and / or carrier. For example, the intended compound may be administered orally as a neutral compound or as a pharmaceutically acceptable salt, or intravenously in a physiological saline solution. Conventional buffers such as phosphates, bicarbonates, or citrates may be used for this purpose. Of course, those skilled in the art can modify the formulation within the scope of the instructions to provide a variety of formulations for a particular route of administration. In particular, the intended compound may be modified to make it more soluble in water or other carriers, for example, this can be readily achieved by minor modifications (salt formulations, esterification, etc.) well known to those skilled in the art. Modifying the route of administration and dosing regimen of a particular compound to control the pharmacokinetics of the compound of the invention to obtain the maximum beneficial effect in the patient is also within the scope of the art.

[0178] Illustrative examples of water-soluble organic solvents used in this method include, but are not limited to, polyethylene glycol (PEG), alcohols, acetonitrile, N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, or combinations thereof. Examples of alcohols include, but are not limited to, methanol, ethanol, isopropanol, glycerol, or propylene glycol.

[0179] Illustrative examples of water-soluble nonionic surfactants used in this method include, but are not limited to, those mentioned above. EL, polyethylene glycol modified (Polyoxyethylene glycerol triricinoleate 35), hydrogenated RH40, hydrogenated RH60, PEG-succinate, polysorbate 20, polysorbate 80 HS (polyethylene glycol 660 12-hydroxystearate), sorbitol monooleate, poloxamer, (ethoxylated persic oil) (Ocyl-hexanoyl polyethylene glycol-8-glyceride) (glycerides) (PEG 6 caprylic / caprylic glyceride), glycerol, diol-polysorbate, or combinations thereof.

[0180] Illustrative examples of water-soluble lipids used in this method include, but are not limited to, vegetable oils, triglycerides, plant oils, or combinations thereof. Examples of lipid oils include, but are not limited to, castor oil, polyoxyethylene castor oil, corn oil, olive oil, cottonseed oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil, hydrogenated soybean oil, triglycerides of coconut oil, palm seed oil, and their hydrogenated forms or combinations thereof.

[0181] Illustrative examples of fatty acids and fatty acid esters used in this method include, but are not limited to, oleic acid, monoglycerides, diglycerides, mono- or di-fatty acid esters of PEG, or combinations thereof.

[0182] Illustrative examples of cyclodextrins used in this method include, but are not limited to, α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, or sulfobutyl ether-β-cyclodextrin.

[0183] Illustrative examples of phospholipids used in this method include, but are not limited to, soybean phosphatidylcholine or distearate phosphatidylglycerol, and their hydrogenated forms, or combinations thereof.

[0184] Delayed-release formulations

[0185] Compounds of formula (I) may be formulated for immediate release and rapid absorption, or may be formulated for delayed release. In some embodiments, compounds are formulated for delayed release using methods and compositions that promote delivery of the active ingredient in the lower gastrointestinal tract after the administered formulation has passed through the stomach. Such methods include known enteric coatings, which slow or prevent the release of compounds of formula (I) or pharmaceutically acceptable salts thereof from the stomach, thereby allowing the active drug to be released primarily in the intestine to enhance direct delivery to tissues most affected by IBD. Some useful methods for delayed-release formulations are described, for example, in B. Singh, Modified-release solid formulations for Colonic Delivery, Recent Patents on Drug Delivery and Formulations 2007, Vol. 1(1), 53-63. Compounds of formula (I) or pharmaceutically acceptable salts thereof may be formulated using methods that reduce dissolution in the stomach and / or increase dissolution and absorption in the lower gastrointestinal (GI) tract to increase the availability of the active drug in the target tissue.

[0186] Delayed release can be achieved by using one or a combination of two or more of the following: pH control (or delayed release) systems, time control (or time-dependent) systems, microbial control systems, and pressure control systems.

[0187] Those skilled in the art can modify formulations within the scope of the teachings of the specification to provide a variety of formulations for specific routes of administration. In particular, compounds can be modified to make them more soluble in water or other carriers. Modifying the route of administration and dosing regimen of specific compounds to control the pharmacokinetics of the compounds of the present invention to obtain maximum beneficial effects in patients is also within the scope of the art.

[0188] Drug combination

[0189] The method of implementation includes administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to a subject requiring treatment for inflammatory bowel disease. The compound of formula (I) may be administered as a neutral compound or as a pharmaceutically acceptable salt. In some embodiments, it is administered as a malate. The compound of formula (I) or a pharmaceutically acceptable salt thereof may be administered as a single agent or in combination with other therapeutic agents. Optionally, the compound of formula (I) or a pharmaceutically acceptable salt thereof may be administered in combination with one or more other therapeutic agents (particularly those known to be used for the treatment of inflammatory bowel disease). These include, but are not limited to:

[0190] a) Anti-TNFα agents (e.g., infliximab, adalimumab, pecelizumab, golimumab);

[0191] b) Sphingosine-1-phosphate (S1P) receptor modulators (e.g., ozamod);

[0192] c) Anti-adhesion (anti-integrin) agents (e.g., nastatinumab, vedotinumab, ertrlizumab);

[0193] d) IL-12 / IL-23 inhibitors (e.g., ustekinumab, risankizumab);

[0194] e) Transforming growth factor β (TGFβ) inhibitors (e.g., Mongersen, Pirfenidone);

[0195] f) Phosphodiesterase 4 (PDE4) inhibitors (e.g., aprestin);

[0196] g) Janus kinase (JAK) / STAT signal transduction and transcription activator inhibitors (e.g., tofacitinib, filagrinib);

[0197] h) Stem cell transplantation (e.g., hematopoietic stem cells, adipose-derived stem cells);

[0198] i) Fecal microbiota transplantation (FMT);

[0199] j) Plasminogen activator inhibitor-1 (PAI-1) inhibitors (e.g., MDI-2268, titraxtinin);

[0200] k) Aminosalicylates (e.g., mesalazine, balsalazine, olsalazine);

[0201] l) Anti-inflammatory corticosteroids; and,

[0202] m) Immune pathway inhibitors, such as azathioprine, mercaptopurine, cyclosporine, and methotrexate.

[0203] The method also includes using a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with other therapies for the treatment of IBD, including treatments such as fecal microbiota transplantation and stem cell transplantation.

[0204] The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with another IBD therapeutic agent or therapy includes co-administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof with another IBD therapeutic agent and concurrent use of another IBD therapeutic agent or therapy in a given patient, wherein the other IBD therapeutic agent or therapy is administered separately from the compound of formula (I) or a pharmaceutically acceptable salt thereof, even if its administration date is different from that of the compound of formula (I), provided that the different treatments are administered in a sequence and time window in which they are expected to provide therapeutic benefit to the subject simultaneously. Therefore, whenever a subject expects to receive IBD therapeutic efficacy from a compound of formula (I) and another IBD therapeutic agent or therapy for any time period, the compound of formula (I) or a pharmaceutically acceptable salt thereof is used in combination with an IBD therapeutic agent or therapy.

[0205] The additional IBD treatment agent may be administered in a pharmaceutical composition separate from the compound of formula (I) or a pharmaceutically acceptable salt thereof, or, when the route of administration and timing of administration of the additional IBD treatment agent and the compound of formula (I) or a pharmaceutically acceptable salt thereof are compatible, the additional IBD treatment agent may be included together with the compound of formula (I) or a pharmaceutically acceptable salt thereof in a single pharmaceutical composition. The additional IBD treatment agent may be administered concurrently with, before, or after the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0206] Methods of using compounds of formula (I) and pharmaceutical compositions thereof

[0207] Given the information available in the art regarding the pharmacokinetic and chemical properties of compounds of formula (I), combined with the information provided herein, the selection of a route of administration and a suitable formulation for administering a compound of formula (I) or a pharmaceutically acceptable salt thereof is within the physician's ordinary skill. The physician will be able to monitor the effectiveness of such treatment and adjust the dosage and frequency of administration using known methods.

[0208] To carry out the method of the present invention, compounds of formula (I) or pharmaceutically acceptable salts thereof and pharmaceutical compositions thereof may be administered orally, parenterally, by inhalation, topically, rectally, nasally, orally, vaginally, via implanted reservoir, or by other means of administration. The term “parentereal” as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0209] In a specific embodiment of the method of the present invention, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered orally. The composition for oral administration can be any orally acceptable dosage form, including but not limited to tablets, capsules, emulsions, aqueous suspensions, dispersants, and solutions. In the case of tablets for oral administration, commonly used carriers include lactose and corn starch. Lubricants, such as magnesium stearate, may also be added. For oral administration in capsule form, useful diluents include lactose and dried corn starch.

[0210] When an aqueous suspension or emulsion of a compound of formula (I) or a pharmaceutically acceptable salt thereof is administered orally, the compound may be suspended or dissolved in an oil phase combined with an emulsifier or suspending agent. If desired, certain sweeteners, flavoring agents, or coloring agents may be added. Nasal aerosols or inhalation compositions may be prepared according to techniques well known in the field of pharmaceutical formulation, and may be prepared as solutions, for example, in saline, using suitable preservatives (e.g., benzyl alcohol), absorption enhancers to improve bioavailability, and / or other solubilizers or dispersants known in the art.

[0211] In a preferred embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof is administered orally in the form of tablets, capsules, soft capsules, or suppositories, typically containing 25 mg to 800 mg of the compound (or its malate). A single dose may be contained in a single dosage form such as a pill or capsule, or a single dose may require two, three, four, or more single dosage forms such as pills or capsules. In some embodiments, a single dosage form, such as a pill, tablet, or capsule, contains an appropriate amount of the compound of formula (I) or its malate for a single dose, such as about 25 mg, or 50 mg, or 75 mg, or 100 mg, or 125 mg, or 150 mg, or 175 mg, or 200 mg, or 225 mg, or 250 mg, or 275 mg, or 300 mg, or 350 mg, or 400 mg, or 450 mg, or 500 mg, or 550 mg, or 600 mg, or 650 mg, or 700 mg, or 750 mg, or 800 mg. In some embodiments, a single pill, tablet, soft capsule, suppository, or capsule containing the adult dose is administered to a subject requiring treatment for IBD at least once daily. In a preferred embodiment, the dose containing the compound of formula (I) is administered twice daily.

[0212] Furthermore, as disclosed herein, the compound of formula (I) or a pharmaceutically acceptable salt thereof may be administered alone or in combination with other therapeutic agents. The combination therapy according to the invention comprises administering at least one dose of the compound of formula (I) or a pharmaceutically acceptable salt thereof and at least one other pharmaceutically active ingredient that can be used to treat IBD. The doses of the compound of formula (I) or a pharmaceutically acceptable salt thereof and the other pharmaceutically active agent may be administered separately or together. The amount and relative timing of administration of the compound of formula (I) or a pharmaceutically acceptable salt thereof and the other pharmaceutically active agent are selected to achieve the desired combined therapeutic effect.

[0213] The following examples are provided to illustrate the biological activity of compounds of formula (I) in colitis model systems, and are not intended to limit the scope of the invention.

[0214] Example

[0215] Example 1. Effect of malate of formula (I) on induced colitis

[0216] Colitis was induced in Wistar rats by intracolonic administration of DNBS. The rats were divided into six groups as described below. Group 1 did not receive DNBS treatment (DNBS- Groups 2-6 were treated with DNBS only on day 1. Group 2 was treated with DNBS without a treatment agent, serving as a disease control. Group 3 was treated with tofacitinib, a known treatment for ulcerative colitis, serving as a positive control. Groups 4 and 5 were treated with different doses of malate of compound (I), and Group 6 was treated with a combination of malate of compound (I) and tofacitinib. Animals were treated daily as described below, starting shortly after administration of DNBS. Treatment lasted for 7 days, during which fecal consistency was monitored. After 7 days, the animals were euthanized, and the weight, length, and ulcer area of ​​the colon in each animal were assessed.

[0217] The test methods and results are summarized below.

[0218] animal

[0219] Animal species and strains: Wistar rats

[0220] Medical history: No treatment received.

[0221] Sex, age, and weight: Male, 5–6 weeks old, 140–160g

[0222] Breeder / Supplier: Shanghai Slack Laboratory Animal Co., Ltd.

[0223] Testing facilities: PharmaLegacy Laboratories Vivarium

[0224] Adaptation period: no less than 7 days

[0225] Room: SPF Room

[0226] Room temperature: 19-26℃

[0227] Room relative humidity: 40-70%

[0228] Light cycle: 12 hours of fluorescent lighting (08:00-20:00) and 12 hours of darkness.

[0229] Animal care: 2-3 rats / cage in the treatment group

[0230] Food: Free access to food (irradiation, Shanghai Slack Laboratory Animal Co., Ltd., China)

[0231] Water: Free access to water (municipal tap water filtered through a water purification system)

[0232] A total of 82 male Wistar rats were obtained from Shanghai Slack Laboratory Animal Co., Ltd. These animals were free of specific pathogens and were approximately 4-5 weeks old upon arrival.

[0233] Assigned to the treatment group

[0234] Animals were randomly assigned to treatment groups using BioBook software to achieve similar group average weights on day 1, controlling for bias.

[0235] Table 1 - Treatment Group

[0236]

[0237]

[0238] a: The solvent for the test sample is distilled water. b: The solvent is 0.5% CMC-Na.

[0239] Wistar rats were induced to have colitis by intracolonic administration of 0.5 mL of DNBS solution (50 mg / mL DNBS dissolved in 30% ethanol) in groups 2-6 on day 1. Meanwhile, group 1 received intracolonic administration of 0.5 mL of 30% ethanol as an ethanol control.

[0240] A total of 82 male Wistar rats were randomly divided into 6 groups as follows:

[0241] Group 1: No treatment received (ethanol control only), N=12

[0242] Group 2: Solvent (DNBS control), N=14

[0243] Group 3: Tofacitinib, 30 MPK, PO, BID, N=14

[0244] Group 4: Malate of Formula I, 30 MPK, PO, QD, N = 14

[0245] Group 5: Malates of Formula I, 100 MPK, PO, QD, N = 14

[0246] Group 6: Malate of Formula I (100 MPK, QD) + Tofacitinib (30 MPK, BID), PO, N = 14

[0247] Record the weight and fecal consistency of all test animals daily. Sacrifice the animals on day 7. Collect each colon. Record the ulcer area, distal colon weight, colon length, and photographs of the relevant colonic region. Divide the colonic tissue longitudinally into three pieces, immediately fixing one piece in 10% neutral buffered formalin. Take two more pieces of colon, flash-freeze in liquid nitrogen, and store at -80°C.

[0248] abbreviation:

[0249] DNBS: 2,4-Dinitrobenzenesulfonic acid

[0250] IBD: Inflammatory Bowel Disease

[0251] CMC-Na: Sodium carboxymethyl cellulose.

[0252] The test sample was prepared as follows: the malate of formula (I) was weighed using an electronic balance, dissolved in distilled water, and then vortexed until fully dissolved.

[0253] As a comparative compound, tofacitinib was included in the test for formula I malate. Tofacitinib is approved for the treatment of rheumatoid arthritis and moderate to severe ulcerative colitis.

[0254] Reference compound: Tofacitinib

[0255] Supplier: Nanjing Yaoshi Technology Co., Ltd.

[0256] Storage conditions: 2~8℃

[0257] Product Number: PBN2011586

[0258] Batch number: PB0000461-169-01

[0259] Prepare a suspension of 3 mg / mL tofacitinib in 0.5% sodium carboxymethyl cellulose: prepare fresh samples twice a week to ensure quality.

[0260] DNBS was dissolved in 30% ethanol to a concentration of 50 mg / mL.

[0261] Reference drug solution: Dilute tofacitinib in 0.5% CMC-Na to a concentration of 3 mg / mL.

[0262] Inducing colitis

[0263] On day 1, animals were randomly divided into 6 groups (see Table 1 for treatment groups) and fasted for 40 hours. For energy intake, 5% glucose saline (10 mL / kg, sc) was provided during the fasting period.

[0264] On day 1 of the study, fasted animals were anesthetized with Zoletil (ip, 25 mg / kg), Zolazepam (ip, 25 mg / kg), and Xylazine (ip, 5 mg / kg).

[0265] For groups 2–6, colitis was induced by intracolonic administration of 0.5 mL DNBS, using a catheter inserted into the colon via the anus up to the splenic flexure (8 cm from the anus). Group 1 received 30% ethanol, also administered intracolonically. Animals exposed to DNBS or ethanol were kept head down for 15 minutes, then maintained in the Trendelenburg position until they awoke to avoid reflux.

[0266] treat

[0267] Group 1: From day 1 to day 7, animals were given distilled water orally 4 hours after taking 30% ethanol, once daily.

[0268] Group 2: From day 1 to day 7, animals were given distilled water orally 4 hours after taking 30% ethanol, once daily.

[0269] Group 3: From day 1 to day 7, animals were given 30 mg / kg (mpk) tofacitinib orally, twice daily, 4 hours after DNBS.

[0270] Groups 4-5: From day 1 to day 7, animals were orally administered different doses of formula I malate, once daily, 4 hours after DNBS.

[0271] Group 6: From day 1 to day 7, 4 hours after DNBS, animals were orally administered 100 mpk of the malate of compound (I) (referred to herein as malate of formula I) qd and 30 mpk of tofacitinib, bid.

[0272] Assessment of colitis

[0273] weight

[0274] Weight was recorded daily throughout the study. The percentage change in weight relative to the starting weight was calculated using the following formula:

[0275] [(Weight on Day X - Initial Weight) / Initial Weight] × 100

[0276] Figure 1 The body weight of the experimental animals during the treatment process was summarized.

[0277] Stool consistency score

[0278] During the experiment, stool samples were monitored daily and their consistency was scored (0 = formed, 1 = moist / viscous, 2 = loose, 3 = liquid) as an indicator of the severity of colitis.

[0279] The above scoring system was used to plot the fecal consistency scores of animals during the 7-day experiment, and the area under the curve (AUC) for each treatment group was calculated using this plot. The AUCs for each treatment group and control group are shown below. Figure 2 As shown.

[0280] Colon weight and length and ulcer area

[0281] On day 7, all animals were euthanized by CO2 asphyxiation followed by cervical dislocation. The abdomen was opened through a midline incision. The contents of the colon were emptied, flushed, and weighed. The length of the colon (from the cecum to the anus) and the ulcer surface area inside the colon were measured. Macroscopic assessments of colon length (CL), colon weight (CW), and ulcer extent (area) were measured in all treatment and control groups, and these results were correlated with CW / CL, CW / BW (body weight), and CW / CL / BW as follows: Figure 3 As shown.

[0282] Note: If the shape of the break is irregular, piece the broken parts together to form a rectangle, and then measure the area of ​​the rectangle (area = length * width).

[0283] Sample collection

[0284] After assessing the length and weight of the colon, the entire colon was divided into three equal parts longitudinally. Two sections of the colon were flash-frozen in liquid nitrogen and stored at -80°C. The third section was fixed in 10% neutral buffered formalin for histopathological evaluation.

[0285] Clinical observation

[0286] Observe the animals daily for signs of disease and general responses to surgery and treatment. Record and detail all abnormalities beyond normal healthy appearance and behavior in the standard PharmaLegacyLaboratories clinical observation form.

[0287] Statistical data

[0288] For body weight, colon length, colon weight, colon weight / length, colon weight / body weight, ulcer area, and other pending parameters, the mean ± SEM was calculated for each group. Statistical analysis was performed using Graphpad Prism, SPSS, or Sigmaplot. Specific statistical tests used are indicated in the legend. Values ​​p < 0.05 were considered statistically significant.

[0289] result

[0290] Compared to the control group treated with only the solvent (ethanol), all groups treated with DNBS showed significant weight loss, increased stool consistency score and AUC, shortened colon length, increased colon weight, and increased ulcer area. This indicates that the model system induced colitis symptoms. The treated groups also had higher CW / CL, CW / BW, and CW / CL / BW ratios compared to the untreated group.

[0291] Tofacitinib was included as a positive control, expected to reduce the effects of colitis, but acting through a different mechanism than the malate of Formula I. In the tofacitinib treatment group receiving 30 mg / kg BID, the CW / CL, CW / BW, and CW / CL / BW ratios were improved by 37%, 9%, and 14%, respectively.

[0292] Animals in the treatment group receiving formula I malate at 30 mg / kg qd showed a significant increase in colon length. Compared to the DNBS-treated control group, the inhibition rates of CW / CL, CW / BW, and CW / CL / BW were increased by 30%, 6%, and 29%, respectively.

[0293] In the animal group treated with 100 mg / kg of formula I malate daily, the CW / CL, CW / BW, and CW / CL / BW ratios improved by 44%, 29%, and 39%, respectively. This indicates that the compound of formula I reduced the degree and / or severity of colitis-induced damage at both doses, and that at higher doses, formula I malate appeared to be more effective than the contrast agent tofacitinib in treating induced colitis.

[0294] Formula I malate in combination with tofacitinib significantly reduced the AUC of the stool consistency score. This suggests that the combination of Formula I malate and tofacitinib may be beneficial for the treatment of IBD.

[0295] Example 2. Effect of malate of formula (I) on TNBS-induced colitis in C57BL / 6 mice.

[0296] Colitis was induced in C57BL / 6 mice by instillation of trinitrobenzenesulfonic acid (TNBS) into the colon, following standard procedures. See Antoniou, et al., Ann. Medicine and Surgery, vol. 11, 9-15 (2016). Mice were then treated with 5-20 mg / kg of formula (I) malate (“SPH-X”) or PBS (control), as shown in each study.

[0297] Macro Observation

[0298] Figure 4A The overall morphology of the colon in mice 7 days after TNBS infusion was shown, comparing the colons of mice treated with twice-daily intraperitoneal delivery of 20 mg / kg SPH-X with those treated with a solvent (PBS). The colons of mice treated with the solvent (PBS) were shorter and swollen, and showed no significant fecal particle formation; this is consistent with the expectations of an ulcerative colitis model. The colons of mice treated with SPH-X (20 mg / kg BID) appeared more normal; they were longer and thinner than those of the solvent-treated mice, and showed significant fecal particles. This suggests that SPH-X treatment treats or prevents the damage that TNBS would cause at the gross physical level in a colitis model.

[0299] Figure 4B The longitudinal anatomy of the TNBS-treated colon is shown to expose the interior of the colon. As mentioned above, the colon treated with TNBS alone exhibited distal hemorrhage, while the colon of animals receiving 10 mg / kg SPH-X intraperitoneally after TNBS infusion did not show this damage. Both external and internal macroscopic observations indicated that TNBS treatment caused obvious, coarse morphological damage consistent with colitis, and that this damage was substantially prevented or reversed by intraperitoneal treatment with SPH-X (malate of formula (I)).

[0300] Microscopic observation

[0301] Figure 5A -B shows the histological observation results of colon sections from C57BL / 6 mice. As described above, all animals were sacrificed on day 3 after TNBS infusion to induce colitis, and the tissues were observed by H&E (hematoxylin and eosin) staining.

[0302] Figure 5A The first image in the diagram shows colon tissue that was infused with only ethanol (without TNBS) and used as a baseline. Figure 5A The second figure shows the colon (colitis control) after TNBS infusion followed by intraperitoneal treatment with PBS twice daily, exhibiting typical tissue damage typical of colitis-induced TNBS-induced colitis.

[0303] Figure 5B The effects of SPH-X at 5 or 10 mg / kg BID in TNBS-infused mice were demonstrated. Figure 5B The first image in the book is colon tissue treated with TNBS infusion and 5 mg / kg SPH-X BID; it shows that this dose of SPH-X can essentially prevent or treat any damage caused by TNBS infusion. Figure 5B The second image shows colon tissue treated with TNBS infusion followed by intraperitoneal administration of 10 mg / kg SPH-X twice daily. It also demonstrates that SPH-X treatment significantly protects the colon from TNBS-induced damage. These images show the microscopic effects of SPH-X in treating or preventing TNBS-induced damage in a mouse model of colitis.

[0304] Biochemical observation

[0305] To assess the role of SPH-X in this model at the molecular level, colitis was induced again in mice via TNBS infusion (three per treatment group), with ethanol as a control. Experimental animals were treated with either the solvent (PBS) or SPH-X (10 mg / kg ip, twice daily) for three days after TNBS injury, followed by sacrifice. Colonic mucosa was isolated from each animal and used for mRNA preparation. mRNA was reverse transcribed to provide cDNA. Cytokines IL-1β and IL-6 were quantified by qRT-PCR. Table 2 shows the data from qRT-PCR, normalized to an ethanol control (no TNBS treatment), with results in... Figure 6 The image is shown in the middle.

[0306] Table 2

[0307]

[0308] SEM 0.412269 5.378737 0.816367 0.437348 4.274689 2.231583

[0309] As expected in colitis models, TNBS infusion resulted in a significant increase in the cytokines IL-1β and IL-6 compared to the ethanol control. Samples from SPH-X mice showed significantly reduced levels of these cytokines. Since colitis damage is thought to be mediated by these (and possibly others) cytokines, this suggests that SPH-X biochemically alleviates TNBS-induced tissue damage and may do so at least in part by reducing cytokine release.

[0310] Finally, mucosal lysates from experimental animals treated as described in the cytokine analysis were prepared and analyzed by Western blotting to understand how SPH-X affects TNF-α protein levels in a colitis model. Figure 7The analysis results are shown; each lane represents one mouse and includes β-actin as a control. No detectable TNF-α protein was observed in the ethanol control animals. In contrast, the TNBS colitis model animals exhibited readily detectable levels of TNF-α. Treatment with 10 mg / kg SPH-X (twice daily for three days; labeled Sph in the figure) reversed this effect of TNBS infusion, as TNF-α protein was not observed in the mucosal isolates from mice treated with SPH-X.

[0311] Example 3. Effects of malate (CFN001 / 01) of formula (I) on human ulcerative colitis and normal mucosal colonic tissue.

[0312] Human colon tissues from healthy donors and donors with ulcerative colitis (UC) were tested under conditions designed to induce cytokine production to determine whether the compound of formula (I) reduced cytokine production in tissues from donors with ulcerative colitis.

[0313] Human ulcerative colitis (UC) and normal gastrointestinal tissue were obtained from surgical remnants of six donors (three normal and three with ulcerative colitis) with informed consent. Donors were pre-screened to exclude subjects who had received any anti-cytokine therapy within the past month. For each sample, smooth muscle was separated from the mucosa and attached to the submucosa. Each sample was dissected using a scalpel to produce 18 full-thickness mucosal biopsies, approximately 5 mm x 5 mm in size. Samples were washed and held in culture medium for approximately 10 minutes while culture plates were prepared.

[0314] Sample preparation

[0315] Staphylococcal enterotoxin B (SEB) (100 μg / mL stock solution) was prepared in phosphate-buffered saline (PBS). The 100 μg / mL stock solution was then diluted in PBS to a concentration of 10 μg / mL, so that 50 μL of this solution was added to 9.95 mL of culture medium to obtain a final well concentration of 50 ng / mL SEB. SEB was added to provide a consistent baseline level for cytokine production.

[0316] Birb 796 in powder form (positive control, Selleck Chemicals catalog number: S1574) was purchased. Birb 796 is a broad-spectrum p38 MAP kinase inhibitor known to inhibit cytokine formation. A 10 mM stock solution was prepared in DMSO. This solution was then added to the culture medium at a volume of 1 μL per 10 mL of medium to achieve an appropriate concentration of 1 μM Birb 796 and 0.01% DMSO.

[0317] CFN001 / 01 (malate of formula (I)) is provided as a powder and prepared as a 10 mM stock solution with distilled water and stored at -20°C. Fresh 10 mM aliquots are used on each experimental day. Working solutions are prepared by diluting the 10 mM aliquots to concentrations of 300, 100, 30, and 10 μM in distilled water.

[0318] Then each working concentration was added to the medium at 1 μl / 1 mL to produce final concentrations of 300, 100, 30 and 10 nM in the wells.

[0319] CMRL medium was prepared using standard methods. Distilled water was used as the solvent and added to the medium at a rate of 1 μl / 1 mL to match the test compound used as a control.

[0320] The sample was placed on the top (mucosal) side of the Netwell filter with the liquid-gas interface facing upwards. The biopsy sample was then incubated at approximately 37°C in a high O2 atmosphere in a medium fortified with an appropriate control or test substance, formula I malate (“CFN001 / 01”). The p38 MAP kinase inhibitor Birb 796 (CAS 285983-48-4) was used as a positive control. Three biopsies were performed for each donor to evaluate each test condition. The test medium for each sample also contained 50 ng / mL Staphylococcus aureus enterotoxin B (SEB) to provide a consistent baseline level of cytokine production. Three biopsies were performed for each donor to evaluate each test condition.

[0321] Test conditions:

[0322] 1. Solvent comparison

[0323] 2. BIRB 796–1μM

[0324] 3.CFN001 / 01–300nM

[0325] 4.CFN001 / 01–100nM

[0326] 5.CFN001 / 01–30nM

[0327] 6.CFN001 / 01–10nM

[0328] Approximately 18 hours after the start of incubation, culture medium samples are collected and rapidly frozen in liquid nitrogen and stored at approximately -80°C until they are ready for ELISA analysis.

[0329] Then, multiplex ELISA was used to analyze IL-1β, IL-17A, TNF-α, IL-6, and IL-23 in the culture medium samples. The multiplex ELISA platform used was Luminex. Luminex, compatible with magnetic bead technology The system was used to quantify each analyte by interpolating a standard curve generated on the same 96-well plate. Each sample was analyzed in duplicate, and the average value was used to plot Figure 8.

[0330] The selected ELISA results are presented graphically in Figure 8, where each plot represents data from one UC subject; this plot shows the effect of CFN001 / 01 on IL-6 release from mucosal tissue samples. Each plot in Figure 8 summarizes the results for a single UC subject, with each point representing cytokine release from one of three replicate tissue samples. The horizontal solid line for each test condition represents the average of the three individual donor means for that test condition, expressed as a percentage of the solvent control.

[0331] like Figure 8A As shown in Figure -C, CFN001 / 01 treatment resulted in a significant dose-related reduction in IL-6 in two of the three subjects, namely donor A ( Figure 8A ) and donor B ( Figure 8B The sample from donor C ( ) had an effect equivalent to or greater than that of the positive control Birb 796. Figure 8C The study showed that Birb-796 (positive control) reduced IL-6, but did not show a reduction in IL-6 with treatment with SPH-X.

[0332] It has been reported that effective treatment of IBD with biological agents is accompanied by a decrease in IL-6 levels, and the decrease in IBD treated with biological IBD agents at week 10 (p = 0.022) was associated with a sustained clinical response after 12 months of treatment. (Caviglia, et al., J. Clin. Med., vol. 9, 800 (2020)). Unresponsive patients did not exhibit lower IL-6 production at week 10, which is the first reported post-treatment test. Therefore, as this report suggests, not all IBD patients respond to all therapies or in the same manner. Similarly, donor C in Figure 8 may be a subject whose UC did not respond to CFN001 / 01, while this subject appears to have responded to Birb 796. In some UC patients, CFN001 / 01 also produced dose-related reductions in the release of the inflammatory cytokines IL-17A and tumor necrosis factor-α (TNF-α).

[0333] The above detailed description is provided to assist those skilled in the art in practicing the invention. However, the scope of the invention described and claimed herein is not limited to the specific embodiments disclosed herein, as these embodiments are intended to illustrate several aspects of the invention. Any equivalent embodiments are intended to be within the scope of the invention. In fact, various modifications to the invention based on the foregoing description, other than those shown and described herein, will become apparent to those skilled in the art without departing from the spirit or scope of the invention. Such modifications are also intended to fall within the scope of the appended claims.

[0334] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes, as if each individual publication, patent, patent application, or other reference were expressly and individually indicated to be incorporated herein by reference in its entirety for all purposes. References cited herein should not be construed as an admission that they are prior art to this invention.

Claims

1. Compound of formula (I) Equation (I) The use of its pharmaceutically acceptable salts in the preparation of medicaments for the treatment of inflammatory bowel disease.

2. The use according to claim 1, wherein, The inflammatory bowel disease mentioned is ulcerative colitis.

3. The use according to claim 1, wherein, The inflammatory bowel disease mentioned is Crohn's disease.

4. The use according to any one of claims 1-3, wherein, The drug is prepared for oral administration or as a suppository.

5. The use according to any one of claims 1-3, wherein, The drug is prepared as a pill, capsule or tablet containing 25 mg to 800 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

6. The use according to any one of claims 1-3, wherein, The drug is prepared as a soft capsule containing 25 mg to 800 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

7. The use according to any one of claims 1-3, wherein, The drug is prepared as an agent that promotes the release of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the lower gastrointestinal tract or reduces its release in the stomach.

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