CN117338784A

CN117338784A - Use of tyrosine kinase inhibitors for the manufacture of a medicament for the treatment of diseases associated with activation of the inflammasome

Info

Publication number: CN117338784A
Application number: CN202311323781.3A
Authority: CN
Inventors: 黄聪龙; 王雅萱
Original assignee: Chang Gung University CGU
Current assignee: Chang Gung University CGU
Priority date: 2020-08-26
Filing date: 2020-08-26
Publication date: 2024-01-05
Also published as: CN114099509B; CN114099509A

Abstract

Disclosed herein is a method for treating a related disease and/or disorder due to an inflammatory body. The method comprises administering to a subject in need thereof an effective amount of 4- [4- [ 5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl ] -carbamoylamino ] -3-fluorophenoxy ] -N-methylpyridine-2-carboxamide, a salt, solvate or ester thereof.

Description

Use of tyrosine kinase inhibitors for the manufacture of a medicament for the treatment of diseases associated with activation of the inflammasome

The patent is 26 days of patent application of 2020, 08 months, application number 2020108720491 and invention creation name: use of tyrosine kinase inhibitors in the manufacture of a medicament for the treatment of diseases associated with activation of the inflammasome

Technical Field

The present invention relates generally to novel uses of tyrosine kinase inhibitors, and in particular to the use of 4- [4- [ 5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl ] -carbamoylamino ] -3-fluorophenoxy ] -N-methylpyridine-2-carboxamide in the treatment of related diseases and/or conditions due to activation of the inflammatory body (inframmamome).

Background

The inflammatory body is an innate immune system receptor that induces inflammation in response to infection and molecules derived from host proteins, and thus has been associated with a variety of autoimmune and autoimmune diseases, including neurodegenerative diseases (e.g., multiple sclerosis, alzheimer's disease, parkinson's disease, etc.), metabolic disorders (e.g., atherosclerosis, type II diabetes, obesity, etc.), and tissue damage, among others. Therefore, agents that inhibit the activation of the inflammatory body should be useful as therapeutic agents for diseases and/or conditions associated with the activation of the inflammatory body.

Disclosure of Invention

The invention provides a novel use of a tyrosine kinase inhibitor 4- [4- [ 5-tertiary butyl-2-quinol-6-yl pyrazol-3-yl ] -carbamoyl amino ] -3-fluorophenoxy ] -N-methylpyridine-2-carboxamide (or 'rebastinib') which is found to be a powerful medicament capable of inhibiting activation of an inflammatory body, and thus rebastinib can be used as a candidate medicament for developing a therapeutic medicament for diseases and/or conditions related to activation of an inflammatory body.

Accordingly, a first aspect of the invention relates to a method of treating an individual suffering from a related disorder caused by activation of an inflammatory body. The method comprises administering to the subject an effective amount of 4- [4- [ 5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl ] -carbamoylamino ] -3-fluorophenoxy ] -N-methylpyridine-2-carboxamide, a salt, solvate or ester thereof.

According to an embodiment of the invention, the related disease caused by activation of the inflammatory body is an autoimmune disease, cancer, infectious disease, inflammatory disease, metabolic disorder, neurodegenerative disease, or tissue damage.

Exemplary autoimmune diseases that can be treated by the methods of the invention include, but are not limited to: ai Disen (Addison's disease), autoimmune thyroid disease, celiac disease, multiple sclerosis, rheumatoid arthritis, psoriasis, type I diabetes, systemic lupus erythematosus (systemic lupus erythematosus, SLE), systemic sclerosis and vitiligo (vitelligo).

Exemplary cancers that may be treated by the methods of the invention include, but are not limited to: b-cell lymphoma, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, gastric cancer, glioblastoma, hodgkin's lymphoma, prostate cancer, and skin cancer.

Exemplary infectious diseases treatable by the methods of the invention include, but are not limited to: bacterial, fungal or viral infections, sepsis and septic shock. In some embodiments, the viral infection is hepatitis B.

Exemplary inflammatory diseases treatable by the methods of the invention include, but are not limited to: atopic dermatitis (actopic dermatitis, AD), asthma, blau syndrome, crohn's disease, cryopyrin-associated periodic syndrome, CAPS), chronic pulmonary obstructive pulmonary disease (chronic obstructive pulmonary disease, COPD), acute respiratory distress syndrome (acute respiratory distress syndrome, ARDS), acute lung injury (acute lung injury, ALI), gout, giant cell arteritis, hepatitis, inflammatory bowel disease, and pulmonary fibrosis.

Exemplary metabolic disorders treatable by the methods of the invention include, but are not limited to: atherosclerosis, arthritis, obesity, and type II diabetes.

Exemplary neurodegenerative diseases treatable by the methods of the invention include, but are not limited to: alzheimer's disease, parkinson's disease, and the like.

Exemplary tissue injuries treatable by the methods of the invention include, but are not limited to: acute liver failure, acute Lung Injury (ALI), ARDS, and the like. Exemplary ALI/ARDS that may be treated by the methods of the present invention include, but are not limited to: transfusion-related lung injury (transfusions-related lung injury), ventilator-induced lung injury (ventilator-induced klung injury), bacterial-induced lung injury, viral-induced lung injury, and the like. In some preferred embodiments, the condition of tissue damage is acute liver failure. In other preferred embodiments, the condition of tissue damage is ALI/ARDS.

According to an embodiment of the invention, the 4- [4- [ 5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl ] -carbamoylamino ] -3-fluorophenoxy ] -N-methylpyridine-2-carboxamide is administered to the individual in an amount of 0.01 to 100 mg/Kg. Preferably, the 4- [4- [ 5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl ] -carbamoylamino ] -3-fluorophenoxy ] -N-methylpyridine 2-carboxamide is administered to the subject in an amount of 0.1 to 80mg/Kg.

According to an embodiment of the invention, the subject suitable for treatment by the method of the invention is a mammal; preferably human beings.

The details of one or more embodiments of the invention are set forth in the accompanying description below. Other features and advantages of the invention will be apparent from the detailed description and from the claims.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and are intended to provide further explanation of the invention claimed.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various exemplary systems, methods, and other exemplary embodiments of various aspects of the invention. The description will be better understood from the following detailed description read with reference to the accompanying drawings in which:

fig. 1: rebastinib inhibits nigericin (nigericin) -induced IL-1 β secretion in Lipopolysaccharide (LPS) -activated d-THP-1 cells. LPS-activated d-THP-1 cells were incubated in DMSO (0.1% as control), rebastinib (0.01-1. Mu.M) or glibenclamide (50M) for 30 min, followed by treatment with nigericin (5. Mu.M) or solvent for 30 min. The cell supernatants were assayed for IL-1β concentration using ELISA kits. Data are shown as mean ± s.e.m. (n=6). P <0.01 compared to control.

Fig. 2: rebamiptinib inhibits monosodium urate (MSU) -induced IL-1 β secretion in LPS-activated d-THP-1 cells. LPS-activated d-THP-1 cells were incubated in DMSO (0.1%, as a control), rebastininib (0.003-1. Mu.M) or glibenclamide (50. Mu.M) for 30 min, and then treated with MSU (100. Mu.g/ml) or solvent for 4 hours. The cell supernatants were assayed for IL-1β concentration using ELISA kits. Data are shown as mean ± s.e.m. (n=6). P <0.05 compared to control; * P <0.01; * P <0.001.

Fig. 3: rebamiptinib inhibits Imiquimod (IMQ) -induced IL-1β secretion in LPS-activated d-THP-1 cells. LPS-activated d-THP-1 cells were incubated in DMSO (0.1%, as a control), rebastininib (0.003-1. Mu.M) or glibenclamide (50. Mu.M) for 30 min, and then treated with IMQ (100. Mu.M) or solvent for 2 hours. The cell supernatants were assayed for IL-1β concentration using ELISA kits. Data are shown as mean ± s.e.m. (n=6). P <0.01 compared to control; * P <0.001.

Fig. 4: rebamiptinib inhibits nigericin-induced secretion of IL-18 in LPS-activated d-THP-1 cells. LPS-activated d-THP-1 cells were incubated in DMSO (0.1% as control), rebastinib (0.01-1. Mu.M) or glibenclamide (50. Mu.M) for 30 min, and then treated with nigericin (5. Mu.M) or solvent for 30 min. The cell supernatants were assayed for IL-18 concentration using ELISA kits. Data are shown as mean ± s.e.m. (n=6). P <0.05 compared to control.

Fig. 5: rebamiptinib does not alter the cell viability of LPS-activated d-THP-1 cells. LPS-activated dTMP-1 cells were incubated in DMSO (0.1%, as control), rebaudilast (0.01-1. Mu.M), glibenclamide (50. Mu.M) or Triton X-100 (0.1%, total lactate dehydrogenase (lactate dehydrogenase, LDH) release) for 1 hour. The cell supernatants were assayed for LDH concentration by CytoTox 96 non-radioactive cytotoxicity assay. Data are shown as mean ± s.e.m. (n=6).

Fig. 6: rebamiptinib inhibits nigericin-induced apoptosis in LPS-activated d-THP-1 cells (pyroptotic cell death). d-THP-1 cells activated by LPS were incubated in DMSO (0.1%), rebastininib (1. Mu.M) or glibenclamide (50. Mu.M) for 30 min, followed by treatment with nigericin (5. Mu.M) or solvent for 30 min. Representative images of cell apoptosis were examined with a scanning electron microscope (n=4).

Fig. 7: rebamiptinib inhibits nigericin-induced focal LDH release in LPS-activated d-THP-1 cells. LPS-activated d-THP-1 cells were incubated in DMSO (0.1% as control), rebauditinib (0.01-1. Mu.M), glibenclamide (50. Mu.M) or Triton X-100 for 30 min, followed by treatment with nigericin (5. Mu.M) or solvent for 30 min. The concentration of LDH in the cell supernatants was analyzed using the CytoTox 96 non-radioactive cytotoxicity assay. Data are shown as mean ± s.e.m. (n=6). P <0.01 compared to control; * P <0.001.

Fig. 8: rebamiptinib inhibits nigericin-induced ASC spot formation in LPS-activated d-THP-1 cells. LPS-activated d-THP-1 cells were incubated in DMSO (0.1% as control), rebastinib (1. Mu.M) or glibenclamide (50. Mu.M) for 30 min, and then treated with nigericin (5. Mu.M) or solvent for 30 min. Cells were fixed with 4% polyoxymethylene (paramldyde) and stained with anti-ASC and Hoechst33342 (n=2). Fluorescence photographs were examined and taken with a fluorescence microscope (IX 81, olympus).

Fig. 9: rebamiptinib can alleviate acute liver injury induced by LPS/D-galactosamine (D-GalN) in mice. The solvent or rebamiptinib (5 or 10 mg/kg) was intravenously administered to 7-10 week old mice (C57 BL/6) for about 1 hour, followed by intraperitoneal injection

LPS (40. Mu.g/kg)/D-GalN (400 mg/kg) was administered to mice for about 5 hours to induce acute liver injury. (A) Representative global images of liver morphology were taken with a digital camera (scale: 1 cm). (B) The weight of the whole liver is expressed as mean ± s.e.m. (n=2-4). * p <0.05; compared with LPS/D-GalN treatment.

Fig. 10: rebamiptinib alleviates serum biochemical parameters in LPS/D-GalN-induced mice. Mice of 7-10 weeks old (C57 BL/6) were treated by intravenous injection with vehicle or rebamiptinib (5 or 10 mg/kg) and then treated with LPS (40. Mu.g/kg)/D-GalN (400 mg/kg) by intraperitoneal injection for further 5 hours, and serum biochemical parameters were determined by an automated clinical chemistry analyzer (Dri-Chem NX500i, fujifilm). (a) AST, (B) ALT, (C) BUN, (D) CRE, and (E) LDH release are expressed as mean ± s.e.m. (n=2-4). P <0.05 compared to LPS/D-GalN alone; * P <0.01.

Fig. 11: rebamiptinib alleviates LPS-induced acute lung injury. Mice (C57 BL/6) of 7-8 weeks old were treated with vehicle or rebastinib (10 mg/kg) by intravenous injection, and after 1 hour, LPS (2 mg/kg in 40. Mu.L of 0.9% physiological saline) was administered by intratracheal spray. After 5 hours, mice were sacrificed and their lung tissues were collected and stained with hematoxylin and eosin (H & E).

Detailed Description

The detailed description provided below in connection with the appended drawings is intended as a description of the present invention and is not intended to represent the only forms in which the present invention may be constructed or utilized.

1. Definition of the definition

The term "salt" refers to a pharmaceutically acceptable salt that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts of the compounds of the invention include acids and bases derived from suitable inorganic and organic acids. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric acids or with organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic or malonic acid, or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, asparaginates, benzenesulfonates, benzoates, bisulphates, borates, butyrates, camphorinates, camphorsulphonates, citrates, cyclopentanepropionates, digluconates, dodecylsulphates, ethanesulphonates, formates, fumarates, glucoheptonates, glycerophosphate, gluconate, hemisulphates, heptanonates, caprates, hydroiodinates, 2-hydroxyethanesulphonates, lactonates, lactates, laurates, maleates, malonates, methanesulfonates, 2-naphthalenesulphonates, nicotinates, nitrates, oleates, oxalates, palmates, hydroxynaphthoates (pamoate), jellates, persulphates, 3-phenylpropionates, phosphates, bitrates, pivalates, propionates, stearates, succinates, sulphates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, and the like. Salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium and N+ (C1-4 alkyl) 4-salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and ammonium cations formed using counterions (e.g., halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate) as appropriate.

The term "solvate" refers to a form of a compound that is associated with a solvent, typically by a solvolysis reaction. This physical association may include hydrogen bonding. Common solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein may be prepared, for example, in crystalline form and may be solvated. Suitable solvates include pharmaceutically acceptable solvates, and further include both stoichiometric and non-stoichiometric solvates. In certain examples, the solvate will be able to isolate when, for example, one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "solvate" encompasses both solution phases and isolatable solvates. Representative solvates include hydrates, ethanolates and methanolates.

The terms "administer," "administering," or "administration" are used interchangeably herein to refer to a mode of delivery, including but not limited to intravenous, intramuscular, intraperitoneal, intraarterial, intracranial, or subcutaneous administration of an agent (e.g., a compound or composition) of the invention. In some embodiments, the compounds of the invention, or salts, solvates thereof, are formulated into tablets for oral administration. In other embodiments, the compounds of the invention, or salts, solvates thereof, are formulated as powders for admixture with a suitable carrier (e.g., buffer solution) prior to use, such as prior to intravenous injection.

An "effective amount" of a compound described herein (taken alone or in combination with another agent) refers to an amount sufficient to elicit a desired biological response, such as inhibiting activation of an inflammatory body or alleviating a disease of interest or symptoms associated with the disease described herein. As will be appreciated by one of ordinary skill in the art, the effective amount of the compounds described herein may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, the age and health of the individual, and the like. In some examples, an effective amount may be a therapeutically effective amount, which refers to an amount of a therapeutic agent sufficient to provide a therapeutic benefit in the treatment of a disorder or to delay onset or minimize one or more symptoms associated with the disorder, alone or in combination with other therapies. The term "therapeutically effective amount" may include an amount that improves overall treatment, reduces or avoids symptoms, signs, or causes of the disorder, and/or enhances the therapeutic efficacy of another therapeutic agent. In other examples, the effective amount may be a prophylactically effective amount. A prophylactically effective amount of a compound refers to an amount of a therapeutic agent used alone or in combination with other drugs that provides a prophylactic benefit in preventing the disorder. For example, a "prophylactically effective amount" of a compound may be an amount sufficient to prevent or delay the onset of, or prevent recurrence of, a disorder or one or more symptoms associated with a disorder. It may also be an amount that improves overall prevention or enhances the prophylactic efficacy of another prophylactic agent.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Also, as used herein, the term "about" generally refers to within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error of the average value when considered by one of ordinary skill in the art. Except in the operating/working examples, or where otherwise explicitly indicated, all numerical ranges, amounts, values, and percentages, for example, numerical ranges, amounts, values, and percentages of materials, amounts, durations, temperatures, operating conditions, ratios of amounts, and the like disclosed herein, are to be understood as modified by the term "about" in any event. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present invention and attached claims are approximations that may vary as desired. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

The singular forms "a," "and," and "the" are used herein to include the plural referents unless the context clearly dictates otherwise.

2. Use of the compounds of the invention

The present invention resides in the unexpected discovery that the tyrosine kinase inhibitor 4- [4- [ 5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl ] -carbamoylamino ] -3-fluorophenoxy ] -N-methylpyridine-2-carboxamide, or rebamiphene, has an inhibitory effect on the irritant of the inflammatory body. Thus, rebamiptinib can be used as a candidate compound for developing a medicament suitable for treating a related disease and/or disorder (e.g., an autoimmune disease, etc.) caused by activation of an inflammatory body.

Accordingly, a first aspect of the present invention provides a method of treating an individual suffering from a disease and/or condition associated with activation of an inflammatory body. The method comprises administering to the subject an effective amount of 4- [4- [ 5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl ] -carbamoylamino ] -3-fluorophenoxy ] -N-methylpyridine-2-carboxamide, a salt, solvate or ester thereof.

The compounds of the present invention may be purchased from commercial sources or may be prepared by any method known in the relevant art. Analysis of the biological activity of the compounds of the present invention shows that they are potent inhibitors of inflammatory body irritants, thus inhibiting pyrolysis and/or ASC spot formation. In addition, the compounds of the present invention are non-cytotoxic and can significantly reduce the level of aspartate Aminotransferase (AST), alanine Aminotransferase (ALT) or Creatinine (CRE) in individuals with acute liver injury. The present invention also demonstrates that the compounds of the present invention are useful as candidates for the development of medicaments suitable for the treatment of diseases and/or conditions associated with activation of the inflammatories.

According to embodiments of the present invention, the disease associated with activation of the inflammatory body may be an autoimmune disease, cancer, infectious disease, inflammatory disease, metabolic disorder, neurodegenerative disease, or a condition of tissue damage.

The autoimmune inflammatory diseases treatable by the methods of the invention include, but are not limited to: ai Disen, autoimmune thyroid disease, celiac disease, multiple sclerosis, psoriasis, rheumatoid arthritis, type I diabetes, SLE, systemic sclerosis and vitiligo.

Exemplary inflammatory diseases treatable by the methods of the invention include, but are not limited to: AD. Asthma, bulaugh syndrome, crohn's disease, CAPS, COPD, ARDS, ALI, gout, giant cell arteritis, hepatitis, inflammatory bowel disease, and pulmonary fibrosis.

Exemplary tissue injuries treatable by the methods of the invention include, but are not limited to: acute liver failure, ALI and ARDS. In some preferred embodiments, the condition of tissue damage is acute liver failure. In other preferred embodiments, the condition of tissue damage is ALI/ARDS, which may be transfusion-associated acute lung injury, ventilator-induced lung injury, bacterial-induced lung injury, or viral-induced lung injury, etc.

According to an embodiment of the present invention, the compound of the present invention, namely 4- [4- [ 5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl ] -carbamoylamino ] -3-fluorophenoxy ] -N-methylpyridine-2-carboxamide, is administered to a subject in an amount of 0.01 to 100mg/Kg, for example 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100mg/Kg; preferably, the 4- [4- [ 5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl ] -carbamoylamino ] -3-fluorophenoxy ] -N-methylpyridine-2-carboxamide is administered to a subject in an amount of 0.1 to 80mg/Kg, for example 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80mg/Kg. In a preferred embodiment, the 4- [4- [ 5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl ] -carbamoylamino ] -3-fluorophenoxy ] -N-methylpyridine-2-carboxamide is administered to a subject in an amount of 0.8 mg/Kg. In another preferred embodiment, the 4- [4- [ 5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl ] -carbamoylamino ] -3-fluorophenoxy ] -N-methylpyridine-2-carboxamide is administered to a subject in an amount of 0.5 mg/Kg. An effective amount of a compound may be administered in one or more doses for one or more days (depending on the mode of administration).

The compounds of the invention may also be formulated with suitable carriers or excipients for suitable routes of administration, for example orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or by implantation in a reservoir. The term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

Suitable dispersing or wetting agents (e.g., TWeen80 And suspending agents to formulate sterile injectable preparations (e.g., sterile injectable aqueous or oleaginous suspensions). The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Among the acceptable vehicle and solvent options, one can use a solution comprising mannitol, water, ringer's solution, and isotonic sodium chloride. In addition, sterile, fixed oils are generally suited as a solvent or suspending medium (e.g., synthetic mono-or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents. Other commonly used surfactants, such as Tweens or Spans or other similar emulsifying agents or bioavailability enhancers, which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms, may also be used for formulation purposes.

Formulations suitable for oral administration may be any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. For oral tablets, common carriers include lactose and corn starch. A lubricant, such as magnesium stearate, is also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the compounds of the invention may be suspended or dissolved in an oily phase in combination with emulsifying or suspending agents. If desired, certain sweeteners, flavoring agents or coloring agents may be added. Nasal aerosol or inhalation formulations may be prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as saline solutions, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons and/or other solubilizing or dispersing agents known in the art. The compounds of the present invention may also be used for rectal administration in the form of suppositories.

Pharmaceutically acceptable carriers or excipients that may be included in formulations containing the compounds of the present invention include inert diluents, solubilizers, dispersing and/or granulating agents, surfactants and/or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring and perfuming agents may also be present in the pharmaceutical composition.

The excipient present in the formulation of the present invention must be "pharmaceutically acceptable" in the sense that it is compatible with the active ingredient of the pharmaceutical composition (and preferably capable of stabilizing the pharmaceutical composition) and not deleterious to the individual to whom the pharmaceutical composition is administered. For example, solubilizing agents that form specific more soluble complexes with the compounds of the invention, such as cyclodextrins, can be employed as pharmaceutically acceptable excipients for delivering the compounds of the invention to a subject. Examples of other pharmaceutically acceptable excipients include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate and D & C yellow No. 10.

Also disclosed herein are kits (e.g., pharmaceutical packages) comprising a compound described herein and a container (e.g., a vial, ampoule, vial, syringe and/or dispenser package, or other suitable container). In some embodiments, the kit may comprise a second container comprising pharmaceutically acceptable excipients for diluting or suspending the formulation of the invention. In some embodiments, the formulations or compounds of the present invention provided in the first container and the second container are combined to form one unit dosage form.

In certain embodiments, the kits described herein are for inhibiting activation of an inflammatory body in a cell. In certain embodiments, the kits described herein are for treating any target disease (e.g., inflammatory disease) as described herein in an individual in need thereof. Thus, any of the kits described herein can comprise instructions for administering a compound or pharmaceutical composition contained therein. Kits of the invention may also contain information required by regulatory authorities such as the FDA. In certain embodiments, the kit and instructions are provided for treating a disease described herein. In certain embodiments, the kit and instructions are provided to prevent the diseases described herein. Kits of the invention may comprise one or more additional agents described herein as separate compositions.

The "subject" to be treated by the methods described herein can be a human subject (e.g., a pediatric subject, such as an infant, child, or adolescent, or an adult subject, such as a young, middle-aged, or elderly person), or a non-human animal, such as a dog, cat, cow, pig, horse, sheep, goat, rodent (e.g., mouse, rat), and non-human primate (e.g., cynomolgus monkey, rhesus monkey). The non-human mammal may be a transgenic or genetically engineered animal. In some examples, the individual is a human patient having, suspected of having, or at risk of having a target disease as described herein (i.e., an autoimmune disease, cancer, infectious disease, inflammatory disease, metabolic disorder, neurodegenerative disease, or a condition secondary to the listed disease). In some embodiments, the individual is a human or non-human mammal having, suspected of having, a condition secondary to inflammatory body activation (e.g., acute liver failure).

Also described herein are methods of inhibiting activation of an intracellular inflammatory body. Such methods comprise contacting a cell with a compound such as described herein in an amount effective to inhibit activation of the inflammatory body. The amount of the compound may be sufficient to inhibit at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%) of the activity of the inflammatory formation stimulator. In some embodiments, the method may be performed in vitro. In other embodiments, they may be performed in vivo by administering the compounds to a subject in need of treatment as described herein.

It is to be understood that a compound or formulation may be used in any of the methods described herein in combination with one or more other agents (e.g., therapeutically and/or prophylactically active agents), as described herein. The compounds or formulations may be administered in combination with other agents that improve their activity (e.g., potency and/or efficacy)), treat the diseases described herein in a subject in need thereof, prevent the diseases described herein in a subject in need thereof, inhibit the activity of an inflammatory body-activated stimulus in a subject or cell, it being further understood that the therapy employed may achieve a desired effect for the same disorder, and/or that it may achieve a different effect.

The invention will now be described more specifically with reference to the following embodiments, which are provided for purposes of illustration and not limitation. Although they are generally those that may be used, other procedures, methods, or techniques known to those of ordinary skill in the art may alternatively be used.

Examples

Materials and methods

Cell culture

Human monocyte strain THP-1 was purchased from American Type Culture Collection (ATCC) and maintained at 37℃in an incubator with 5% CO2 in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS), 1% antibiotic antifungal solution, 0.1% beta-mercaptoethanol (beta-ME) and a phenol red indicator. The medium was changed every 2-3 days.

Induction of macrophage-like cells from THP-1 cells

To induce differentiation, THP-1 cells (cell density about 5X 105/mL) were incubated in serum-free medium containing 100nM phorbol 12-myristate 13-acetate (PMA) for about 4 hours. Then, the culture medium containing PMA was replaced with fresh RPMI-1640 medium, and the culture was continued for 2 days to differentiate THP-1 cells into macrophage-like cells, hereinafter referred to as "d-THP-1 cells".

Inducing production of inflammasome and release of IL-1 beta and IL-18

d-THP1 cells were treated with 100ng/mL Lipopolysaccharide (LPS) for 3 hours, then replaced with fresh culture without LPS for further incubation for 30 minutes, then treated with the test drug for 30 minutes, and finally various stimuli (i.e., nigericin, monosodium urate (MSU) and Imiquimod (IMQ)) were added to the medium to induce the formation of inflammatory bodies, and release IL-1 beta and IL-18, respectively.

Determination of IL-1 beta and IL-18 concentration

The concentrations of IL-1β and IL18 were independently measured using ELISA detection kits (R & D systems) according to the measurement methods provided by the manufacturer. The concentrations of IL-1β and IL-18 were determined by measuring the absorbance at 450nm and 540nm, respectively, using a Multiskan GO multi-plate reader.

Cytotoxicity detection

Cytotoxicity was determined by measuring Lactate Dehydrogenase (LDH), a cytoplasmic enzyme that is released only upon cell death. Briefly, supernatants from d-THP-1 cells with or without treatment with the test compound or with Triton X-100 (0.1% total LDH release) were collected. LDH matrix was added to a tube containing cell supernatant, the mixture was reacted in the dark for 30 minutes, and then the absorbance at 490nm was measured. Cytotoxicity was determined from the percentage of LDH released/total amount of LDH in the cell.

Fluorescent image

d-THP-1 cells (treated with LPS, test compound and stimulus or untreated as described above) were fixed with 4% polyoxymethylene, washed 3 times with PBS, treated with Triton X-100 (0.1%) for 10 min, washed 3 times with PBS again, treated with 5% bsa for 1 hour, and then washed 3 times with PBS again added primary antibody (anti-ASC). Cells labeled with primary antibody (anti-ASC antibody) were incubated overnight at 4 ℃ and then labeled with secondary antibody (the label was performed for 1 hour at room temperature), washed again 3 times with PBS, and after addition of Hoechst33342, photographed using a fluorescence microscope (IX 81 Olympus with Lumen 200 fluorescent illumination system).

Observation of cell apoptosis using field emission scanning electron microscopy

d-THP-1 cells (treated with LPS, test compound and stimulus or untreated as described above) were fixed with 4% polyoxymethylene and 2.5% glutaraldehyde (glutalaldehydes) in order for 10 minutes, washed with PBS, and then reacted with osmium tetroxide (OsO 4) for 30 minutes, respectively; after thoroughly washing the cells with distilled water, they were incubated with tannic acid (tannic acid) for 30 minutes. The cells were dehydrated by a gradient of ethanol, where the concentration of ethanol was gradually increased from 30% to 50%, 70%, 80%, 90% and 100%. The 100% ethanol dehydration step was repeated 3 times. The samples were then dried under supercritical conditions using a critical point dryer (Critical Point Dryer (CPD)) (LEICAME CPD) with a mixture of CO2 and ethanol. The dried samples were then mounted on an alumina stage and sputtered with gold ions (ion sputter, JEOL JFC-1100E, operating at 1-2mA for 60 seconds) and then observed under a Field Emission Scanning Electron Microscope (FESEM).

Animals

C57BL/6 mice (7 to 10 weeks old, each weighing about 20-25 g) were fed a standard laboratory diet in mini-isolator units. Animals were housed under humidity and temperature controlled conditions, the light/dark cycle was set at 12 hour intervals, and maintained under prescribed and inorganic pathogen conditions. All animal studies were performed according to the protocol approved by the university of long heptyl animal care and use committee (taibei, taiwan).

Animal model for acute liver failure

Mice were randomly divided into 4 groups. Test compound (5 or 10 mg/Kg) or control solvent (80% saline, 10% ethanol, 10% Tween-20) was injected by tail vein of each mouse. Mice were allowed to rest for 1 hour, then were challenged with acute liver failure by intraperitoneal injection of LPS (40. Mu.g/ml)/D-GalN (400 mg/kg). After 5 hours the animals were sacrificed and blood samples and tissue samples were collected, respectively. Blood samples were analyzed for levels of LDH, AST, ALT, BUN and CRE, respectively. Liver tissue was fixed with 10% formalin (formalin), sectioned, and stained with hematoxylin and eosin (H & E).

Animal model of Acute Lung Injury (ALI)

ALI production was induced by applying LPS (2 mg/kg) to 7-8 week old mice (C57 BL/6) by intratracheal spraying. After overnight fasted mice were injected with rebaudinib (10 mg/kg) or an equivalent amount of solvent (vehicle) by intravenous injection. After 1 hour, LPS (2 mg/kg in 40. Mu.L of 0.9% saline) or 0.9% saline was administered by intratracheal spray. After 5 hours, mice were sacrificed and their lung tissues were collected, fixed with 10% formalin (formalin), sectioned, and stained with hematoxylin and eosin (H & E).

EXAMPLE 1 Compounds of the invention inhibit Nigericin-induced IL-1 beta secretion in LPS-activated d-THP-1 cells

In this example, the effect of the compounds of the invention (i.e., rebastinib) on the release of IL-1β from LPS-activated macrophage-like cells (i.e., d-THP-1 cells) was investigated. To this end, d-THP-1 cells were first activated with LPS (100 ng/mL), then treated with various concentrations (i.e., 0.01, 0.03, 0.1, 0.3 or 1. Mu.M) of the present compounds or glibenclamide (50. Mu.M, positive control), and finally stimulated with a stimulus (i.e., nigericin (5. Mu.M), MSU (100. Mu.g/mL) or IMQ (100. Mu.M)), and the results are shown in FIGS. 1 to 3.

As shown, nigericin resulted in significant amounts of IL-1β release from LPS-activated d-THP-1 cells, which release could be reduced by the compounds of the invention (FIG. 1). Similarly, MSU and IMQ also result in the release of large amounts of IL-1β, which can be inhibited by the compounds of the present invention (FIGS. 2 and 3), respectively.

EXAMPLE 2 Compounds of the invention inhibit Nigericin-induced IL18 secretion in LPS-activated d-THP-1 cells

In this example, the effect of the compounds of the invention (i.e., rebastinib) on the release of IL-18 from LPS-activated macrophage-like cells (i.e., d-THP-1 cells) was investigated. For this purpose, d-THP-1 cells were first activated with LPS (100 ng/ml), then treated with various concentrations (i.e. 0.01, 0.03, 0.1, 0.3 or 1. Mu.M) of the compound of the invention or glibenclamide (50. Mu.M, positive control) and finally stimulated with nigericin (5. Mu.M), the results are shown in FIG. 4.

As shown, nigericin resulted in a significant amount of IL-18 release from LPS-activated d-THP-1 cells, which was significantly reduced by the compounds of the invention (FIG. 4).

EXAMPLE 3 cytotoxicity Studies of Compounds of the invention

In this example, the cytotoxic effect of the compounds of the invention was studied by measuring LDH. The results are shown in FIG. 5.

As shown, the compounds of the present invention are not cytotoxic to the tested d-THP-1 cells themselves.

EXAMPLE 4 Compounds of the invention inhibit Nigericin-induced apoptosis and Coke-apoptotic LDH release in LPS-activated d-THP-1 cells

Pyrosis (pyrosis) is a form of programmed cell death. In the process, immune cells recognize foreign danger signals within themselves, release pro-inflammatory cytokines, swell, burst, and then die. In this example, the effect of the compounds of the invention on nigericin-induced apoptosis and LDH release was studied, wherein the phenomenon of apoptosis was observed using a field emission scanning electron microscope and LDH levels were determined by enzymatic assays.

Referring to FIG. 6, a photograph of Nigericin stimulated LPS-activated d-THP-1 cells in the presence or absence of a compound of the invention. As expected, nigericin induced swelling and lysis of LPS-activated d-THP-1 cells, but significantly reduced cell apoptosis when pretreated with the compounds of the invention as well as glibenclamide (positive control). Furthermore, the compounds of the invention were also observed to inhibit LDH release (fig. 7).

EXAMPLE 5 Compounds of the invention inhibit Nigericin-induced ASC Spot formation in LPS-activated d-THP-1 cells

The hallmark of inflammatory body activation is the formation of ASC spots, which are microscale structures formed by the inflammatory body adapter protein ASC (CARD-containing apoptosis-related spot-like protein) consisting of a thermo-protein domain (PYD) and a cysteine protease recruitment domain (caspase recruitment domain (CARD)). In this example, the effect of the compounds of the invention on LPS-activated d-THP-1 cells was observed using a fluorescence microscope and the results are shown in FIG. 8.

As is clear from the photograph in fig. 8, significant ASC spots formed after treatment with nigericin, but the formation of ASC spots was greatly reduced if the cells were pretreated with the compound of the present invention or glibenclamide (positive control). The data of this example clearly show that the compounds of the invention inhibit the inflammatory body so that it is not activated.

EXAMPLE 6 Compounds of the invention alleviate LPS/D-GalN-induced acute liver injury in mice

An animal model of acute liver injury was used in this example to study the effect of the compounds of the invention on acute liver failure. Briefly, animals were first treated with LPS/D-GalN (LPS: 40. Mu.g/mL; D-GalN:400 mg/mL) to cause acute liver injury, and then blood and tissue samples were taken before and after treatment with the compounds of the present invention, respectively, and then subjected to biochemical and microscopic analysis. The results are shown in fig. 9 and 10.

As is evident from fig. 9, LPS/D-GalN treatment caused liver damage, since the overall image of damaged liver tissue was dark red, which is an indication of tissue bleeding (fig. 9, panel a). In contrast, images taken from liver samples of animals treated with compounds of the present invention showed a significant decrease in bleeding and inflammation, which was also reflected in a decrease in tissue weight (fig. 9, panel b). Furthermore, after treatment with the compounds of the invention, various indicators of liver tissue damage, including ALT, AST, CRE and LDH, each level was significantly reduced. EXAMPLE 7 Compounds of the invention alleviate LPSN-induced Acute Lung Injury (ALI) in mice

Animal models of ALI were used in this example to study the effect of the compounds of the invention on ALI. Briefly, animals 7-8 weeks old were treated with LPS (2 mg/Kg) by intratracheal spraying to cause ALI. Mice were fasted overnight, injected intravenously with rebastinib (10 mg/kg) or an equivalent amount of solvent, and after 1 hour, animals were given ALI by intratracheal spraying with LPS (2 mg/kg in 40 μl of 0.9% saline) or 0.9% saline. Mice were sacrificed after 5 hours and their lung tissue was taken. Tissues were fixed with 10% formalin and then stained with H & E. The results are shown in FIG. 11.

From the photograph of fig. 11, it is shown that the solvent treated lung tissue does not exhibit ALI; in contrast, LPS-treated lung tissue has altered cell morphology and infiltration. In the case of treatment with rebaudinib (10 mg/kg) prior to the induction of ALI with LPS, the degree of change in the lung tissue pattern or infiltration due to LPS was significantly improved. This result shows that rebamiptinib can improve or cure ALI.

In general, the compounds of the invention inhibit the activation of the inflammasome and thus may be used as candidates for the development of medicaments for the treatment of diseases and/or disorders associated with the activation of the inflammasome, including disorders secondary to the diseases and/or disorders associated with the activation of the inflammasome, such as acute liver failure and ALI.

It will be appreciated that the description of the above embodiments is given by way of example only and that various modifications may be made by persons skilled in the art. The above specification, examples and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of this invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those skilled in the art could make numerous alterations to the disclosed embodiments without departing from the scope of this invention.

Claims

1. Use of 4- [4- [ 5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl ] -carbamoylamino ] -3-fluorophenoxy ] -N-methylpyridine-2-carboxamide with a pharmaceutically acceptable carrier or excipient for the manufacture of a medicament for use in the inhibition of inflammatory small body IL-1β, IL-18 release or pyropheoplastic LDH release.