TWI769506B - Methods for the treatment of diseases associated with activation of inflammasomes - Google Patents

Abstract

Disclosed herein is a method of treating diseases and/or disorders associated with the activation of inflammasomes. The method includes 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, a solvate or an ester thereof.

Description

Methods for treating diseases associated with activation of inflammasomes

The present disclosure relates generally to new uses of tyrosine kinase inhibitors, particularly 4-[4-[5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl]-carbamide Use of acylamino]-3-fluorophenoxy]-N-picoline-2-carboxamide for the treatment of diseases and/or conditions associated with inflammasome activation.

Inflammasomes are receptors of the innate immune system that induce inflammation in response to infection and molecules derived from host proteins, and have therefore been associated with a variety of autoinflammatory and autoimmune diseases, including neurodegenerative diseases (eg, multiple sclerosis, Alzheimer's disease (Alzheimer's disease), Parkinson's disease (Parkinson's disease, etc.), metabolic disorders (such as atherosclerosis, type II diabetes, obesity, etc.) and tissue damage. Therefore, agents that inhibit the activation of inflammasomes should be useful as therapeutic drugs for related diseases and/or disorders caused by inflammasome activation.

The present disclosure provides a tyrosine kinase inhibitor, 4-[4-[5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl]-aminocarbamoylamino]-3-fluoro A novel use of phenoxy]-N-picoline-2-carboxamide (or "rebastinib"), which was found to be a potent agent that inhibits inflammasome activation, therefore, Basitinib is a drug candidate for the development of therapeutics for diseases and/or conditions associated with activation of the inflammasome.

Accordingly, a first aspect of the present disclosure relates to a method of treating an individual suffering from a disease associated with inflammasome activation. The method comprises administering to the individual an effective amount of 4-[4-[5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl]-aminocarbamoylamino]-3- Fluorophenoxy]-N-picoline-2-carboxamide, its salts, solvates or esters.

According to embodiments of the present disclosure, the associated disease due to inflammasome activation is an autoinflammatory disease, cancer, infectious disease, inflammatory disease, metabolic disorder, neurodegenerative disease, or tissue damage.

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

Exemplary cancers that can be treated by the methods of the present 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 that can be treated by the methods of the present 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 that can be treated by the methods of the present invention include, but are not limited to: atopic dermatitis (AD), asthma, Blau syndrome, Crohn's disease, cryptic fever-related Cryopyrin-associated periodic syndrome (CAPS), chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), acute lung injury (ALI) , gout, giant cell arteritis, hepatitis, inflammatory bowel disease, and pulmonary fibrosis.

Exemplary metabolic disorders that can be treated by the methods of the present invention include, but are not limited to, atherosclerosis, arthritis, obesity, and type II diabetes.

Exemplary neurodegenerative diseases that can be treated by the methods of the present invention include, but are not limited to, Alzheimer's disease, Parkinson's disease, and the like.

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

According to an embodiment of the present disclosure, the 4-[4-[5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl]-aminocarbamoylamino]-3-fluorobenzene Oxy]-N-picoline-2-carboxamide is administered to the subject in an amount of 0.01 to 100 mg/Kg. Preferably, the 4-[4-[5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl]-aminocarbamoylamino]-3-fluorophenoxy]- N-picoline-2-carboxamide is administered to the subject in an amount of 0.1 to 80 mg/Kg.

According to embodiments of the present disclosure, individuals suitable for treatment by the methods of the present invention are mammals; preferably humans.

The details of one or more implementations of the present disclosure are set forth in the description below. Other features and advantages of the present invention will become apparent from the detailed description and the scope of the patent application.

It is to be understood that both the foregoing general description and the following detailed description have been given by way of example and are intended to provide further explanation of the claimed invention.

The detailed description provided below in connection with the appended drawings is intended as an illustration of the present disclosure and is not intended to represent the only form in which the present disclosure may be constructed or utilized

1. definition

The term "salt" refers to a pharmaceutically acceptable salt which, within the scope of sound medical judgment, is suitable for use in contact with human and lower animal tissues without undue toxicity, irritation, allergic reaction, etc., and which is compatible with reasonable commensurate with the benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are amine groups with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric acids or with organic acids such as acetic, oxalic, maleic, tartaric, citric, Salts formed from succinic acid or malonic acid, or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, besylate, benzoate, bisulfate, borate, butyrate, camphorate , camphorsulfonate, citrate, cyclopentane propionate, digluconate, lauryl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerin Phosphate, Gluconate, Hemisulfate, Heptanoate, Caproate, Hydroiodide, 2-Hydroxyethanesulfonate, Lactobate, Lactate, Laurate, Lauryl Sulfate, Malic Acid salt, maleate, malonate, mesylate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate ), jelly, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, sulfur Cyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium and N ^{+ (} _{C1-4alkyl )4 - salts} . Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium and the use of counter ions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and arylsulfonate) amine cations.

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

The terms "administration," "administration," or "administration" are used interchangeably herein to refer to modes of delivery including, but not limited to, intravenous, intramuscular, intraperitoneal, intraarterial, intracranial, or subcutaneous administration of an agent of the invention (eg, a compound or composition). In some embodiments, the compounds of the present disclosure, 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 (eg, a buffered solution) prior to use, eg, intravenous injection.

An "effective amount" of a compound described herein (administered alone or in combination with another agent) refers to an amount sufficient to elicit a desired biological response, such as inhibiting inflammasome activation or alleviating a target disease or symptoms associated with the disease described herein. As will be understood by those of ordinary skill in the art to which this invention pertains, 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 of the individual, and factors such as health status. In some instances, an effective amount can 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 the onset or minimize the onset of one or more symptoms associated with the disorder, alone or In combination with other therapies. The term "therapeutically effective amount" can 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 can be a prophylactically effective amount. A prophylactically effective amount of a compound refers to the amount of a therapeutic agent, 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 can be an amount sufficient to prevent or delay the onset or prevent the recurrence of the disorder or one or more symptoms associated with the disorder. It can also be an amount that improves overall prophylaxis 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 contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Also, as used herein, the term "about" generally means 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 mean when considered by one of ordinary skill in the art to which this invention pertains. Except in operating/working examples, or unless expressly stated otherwise, all numerical ranges, amounts, values and percentages, such as numerical ranges for amounts of materials, durations, temperatures, operating conditions, ratios of amounts, etc. disclosed herein, Amounts, values and percentages, in any event, should be understood to be modified by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and the appended claims are approximations that can vary as desired. At a minimum, each numerical parameter should at least be interpreted in light of the number of reported significant digits and applying ordinary rounding techniques.

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

2. Use of the compounds of the present invention

The present disclosure resides in the unexpected discovery that the tyrosine kinase inhibitor 4-[4-[5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl]-aminocarbamoylamino]- 3-Fluorophenoxy]-N-picoline-2-carboxamide) (or ribasitinib) has inhibitory effects on inflammasome stimulants. Therefore, ribasitinib can be used as a candidate compound to develop drugs suitable for the treatment of related diseases and/or disorders (eg, auto-inflammatory diseases, autoimmune diseases, etc.) due to inflammasome activation.

Accordingly, a first aspect of the present disclosure is to provide a method of treating an individual suffering from a disease and/or disorder associated with inflammasome activation. The method comprises administering to the individual an effective amount of 4-[4-[5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl]-aminocarbamoylamino]-3- Fluorophenoxy]-N-picoline-2-carboxamide, its salts, solvates or esters.

The compounds of the present invention can be purchased from commercial sources or can be prepared by any method known in the relevant art. Bioactivity assays of the compounds of the present invention show that they are potent inhibitors of inflammasome stimulators, thereby inhibiting pyrolysis and/or ASC spot formation. Furthermore, the compounds of the present invention are not cytotoxic and significantly reduce aspartate aminotransferase (AST), alanine aminotransferase (ALT) or creatinine (CRE) in individuals with acute liver injury s level. The present disclosure also demonstrates that the compounds of the present invention may be candidates for the development of drugs suitable for the treatment of diseases and/or disorders associated with inflammasome activation.

According to embodiments of the present disclosure, the associated disease due to inflammasome activation may be an autoinflammatory disease, cancer, infectious disease, inflammatory disease, metabolic disorder, neurodegenerative disease, or a disorder of tissue damage.

Sexual autoinflammatory diseases treatable by the methods of the present invention include, but are not limited to: Addison's disease, autoimmune thyroid disease, celiac disease, multiple sclerosis, psoriasis, rheumatoid arthritis, type I diabetes, SLE, systemic sclerosis, and vitiligo.

Exemplary cancers that can be treated by the methods of the present 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 that can be treated by the methods of the present 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 that can be treated by the methods of the present invention include, but are not limited to: AD, asthma, Blau syndrome, Crohn's disease, CAPS, COPD, ARDS, ALI, gout, giant cell arteritis, hepatitis, inflammatory bowel disease, and pulmonary fibrosis.

Exemplary metabolic disorders that can be treated by the methods of the present invention include, but are not limited to, atherosclerosis, arthritis, obesity, and type II diabetes.

Exemplary neurodegenerative diseases that can be treated by the methods of the present invention include, but are not limited to, Alzheimer's disease, Parkinson's disease, and the like.

Exemplary tissue injuries that can be treated by the methods of the present invention include, but are not limited to, acute liver failure, ALI, and ARDS. In some preferred embodiments, the tissue damaging condition is acute liver failure. In other preferred embodiments, the tissue damage condition is ALI/ARDS, which can be blood transfusion-related acute lung injury, respirator-induced lung injury, bacteria-induced lung injury, or virus-induced lung injury, and the like.

According to an embodiment of the present disclosure, a compound of the present disclosure, namely 4-[4-[5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl]-aminocarbamoylamine [methyl]-3-fluorophenoxy]-N-picoline-2-carboxamide, is administered to the subject in an amount of 0.01 to 100 mg/Kg, such as 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 100 mg/Kg; preferably, the 4-[4-[5-tert-butyl-2-quinoline- 6-ylpyrazol-3-yl]-aminocarbamoylamino]-3-fluorophenoxy]-N-picoline-2-carboxamide is administered in an amount of 0.1 to 80 mg/Kg Individual, eg 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 mg/Kg. In a preferred embodiment, the 4-[4-[5-tert-butyl-2-quinolin-6-ylpyrazol-3-yl]-aminocarbamoylamino]-3-fluorobenzene Oxy]-N-picoline-2-carboxamide was administered to the 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]-aminocarbamoylamino]-3-fluoro Phenoxy]-N-picoline-2-carboxamide is administered to subjects in an amount of 0.5 mg/Kg. An effective amount of the compound can be administered in one or more doses for one or several days (depending on the mode of administration).

The compounds of the present invention may also be formulated with suitable carriers or excipients for a suitable route of administration, eg, oral, parenteral, by inhalation spray, topical, rectal, nasal, buccal, vaginal or by implantation 's repository. The term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

Sterile injectable preparations (eg, sterile injectable aqueous or oleaginous suspensions) can be formulated according to techniques known in the art using suitable dispersing or wetting agents (eg, Tween® 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the options of acceptable vehicles and solvents that can be used include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are usually also suitable as a solvent or suspending medium (eg, synthetic mono- or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives, are used 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. For formulation purposes, 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.

Formulations suitable for oral administration can 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 cornstarch. Lubricants such as magnesium stearate are also often added. For oral administration in capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions or emulsions are administered orally, the compounds of the present disclosure can be suspended or dissolved in an oily phase in combination with an emulsifying or suspending agent. If desired, certain sweetening, flavoring or coloring agents may be added. Nasal aerosol or inhalation formulations may be prepared according to techniques well known in the pharmaceutical formulation arts and may be prepared as saline solutions using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons and /or other solubilizers or dispersants known in the art. The compounds of the present disclosure may also be used for rectal administration in the form of suppositories.

Pharmaceutically acceptable carriers or excipients that can be included in formulations containing compounds of the present disclosure include, inert diluents, solubilizers, dispersing and/or granulating agents, surfactants and/or emulsifying agents, 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 can also be present in the pharmaceutical compositions.

An excipient present in the formulation of the present invention must be "pharmaceutically acceptable", meaning that the excipient is compatible with the active ingredients of the pharmaceutical composition (and preferably is capable of stabilizing the pharmaceutical composition) and is The individual to whom the pharmaceutical composition is administered is harmless. For example, solubilizers, such as cyclodextrins, which form specific more soluble complexes with the compounds of the present invention, can be used as pharmaceutically acceptable excipients for delivering the compounds of the present invention into a subject. Examples of other pharmaceutically acceptable excipients include colloidal silica, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow No. 10.

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

In certain embodiments, the kit lines described herein are used to inhibit inflammasome activation in a cell. In certain embodiments, the kits described herein are used to treat any of the target diseases (eg, inflammatory diseases) as described herein in an individual in need thereof. Accordingly, any of the kits described herein can include instructions for administering the compound or pharmaceutical composition contained therein. The kits of the present invention may also contain information required by regulatory agencies such as the FDA. In certain embodiments, the kits and instructions are provided to treat the diseases described herein. In certain embodiments, the kits and instructions are provided to prevent the diseases described herein. The kits of the present invention may comprise one or more of the additional agents described herein as separate compositions.

An "individual" to be treated by the methods described herein can be a human individual (eg, a pediatric individual, such as an infant, child, or adolescent, or an adult individual, such as a young, middle-aged, or elderly person), or a non-human animal, such as Dogs, cats, cows, pigs, horses, sheep, goats, rodents (eg, mice, rats) and non-human primates (eg, cynomolgus monkeys, rhesus monkeys). The non-human mammal can be a transgenic animal or a genetically engineered animal. In some examples, the individual is suffering from a target disease as described herein (ie, auto-inflammatory disease, cancer, infectious disease, inflammatory disease, metabolic disorder, neurodegenerative disease, or a disorder secondary to the listed diseases) , human patients suspected of having, or at risk for, the disease. In some embodiments, the individual is a human or non-human mammal having, suspected of having, a disorder secondary to inflammasome activation (eg, acute liver failure).

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

It is understood that, as described herein, a compound or formulation can be used in any of the methods described herein in combination with one or more other agents (eg, therapeutically and/or prophylactically active agents). The compounds or formulations can be administered in combination with other agents that improve their activity (eg, activity (eg, potency and/or efficacy)) for the treatment of the diseases described herein in individuals in need thereof, the prevention of the diseases described herein in individuals in need thereof disease, inhibition of the activity of stimulators of inflammasome activation in an individual or cell, it is also understood that the therapy employed may achieve the desired effect for the same condition, and/or it may achieve different effects.

The present invention will now be described in more detail 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 to which this invention pertains may alternatively be used.

Example

Materials and Methods

cell culture

The human monocytic cell line THP-1 was purchased from the American Type Culture Collection (ATCC) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine in a 37°C, 5% _CO2 incubator Serum (FBS), 1% antibiotic-antifungal solution, 0.1% β-mercaptoethanol (β-ME), and phenol red indicator. The medium was changed every 2-3 days.

from THP-1 Induction of macrophage-like cells (macrophage-like cells)

To induce differentiation, THP-1 cells (at a cell density of about 5x10 ⁵ /mL) were grown in PBS containing 100 nM phorbol 12-myristate 13-acetate (PMA). in serum-free medium for approximately 4 hours. Then, the medium containing PMA was replaced with fresh RPMI-1640 medium, and the culture was continued for another 2 days to differentiate the THP-1 cells into macrophage-like cells, hereinafter referred to as "d-THP-1 cells".

Inflammasome production and release IL-1β and IL-18

d-THP1 cells were first treated with 100 ng/mL lipopolysaccharide (LPS) for 3 hours, then replaced with fresh culture without LPS for 30 minutes, then added with test drugs for 30 minutes, and finally added various stimuli to the medium. (ie, nigericin, monosodium urate (MSU), and imiquimod (IMQ)) to induce inflammasome formation and release IL-1β and IL-18.

Determination IL-1β and IL-18 concentration

Concentrations of IL-1β and IL-18 were independently determined with an ELISA assay kit (R&D Systems) according to the assay method provided by the manufacturer. The concentrations of IL-1β and IL-18 were determined by measuring the absorbance at 450 nm and 540 nm, respectively, on a Multiskan GO multiplate reader.

Cytotoxicity assay

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

fluorescent image

d-THP-1 cells (treated or untreated with LPS, test compounds and stimuli as described above) were fixed with 4% paraformaldehyde, washed 3 times with PBS and treated with Triton X-100 (0.1%) for 10 min, washed 3 times with PBS again, treated with 5% BSA for 1 hour, then washed 3 times with PBS before adding primary antibody (anti-ASC). Cells labeled with primary antibody (anti-ASC antibody) were incubated overnight at 4°C, then labeled with secondary antibody (the labeling was performed at room temperature for 1 hour), washed 3 times with PBS, and after adding Hoechst 33342 , photographed using a fluorescence microscope (IX81 Olympus, with Lumen 200 fluorescence illumination system).

Observation of Pyroptosis Using Field Emission Scanning Electron Microscopy

d-THP-1 cells (treated or untreated with LPS, test compounds and stimuli as described above) were sequentially fixed with 4% paraformaldehyde and 2.5% glutaraldehyde for 10 min, washed with PBS, It was then reacted with osmium tetroxide (OsO ₄ ) for 30 minutes; after the cells were thoroughly washed with distilled water, they were incubated with tannic acid for 30 minutes. The cells were dehydrated by a gradient solution of ethanol, with the concentration of the ethanol gradually increasing from 30% to 50%, 70%, 80%, 90% and 100%. Repeat the 100% ethanol dehydration step 3 times. The samples were then dried with a mixture of CO ₂ and ethanol under supercritical conditions using a Critical Point Dryer (CPD) (LEICAME CPD 300). The dried samples were subsequently mounted on an alumina platform and sputtered with gold ions (ion sputter, JEOL JFC-1100E, operating at 1-2 mA for 60 s), followed by field emission scanning electron microscopy (FESEM) observe below.

animal

C57BL/6 mice (7 to 10 weeks old, each weighing approximately 20–25 g) were housed in microisolator units on a standard laboratory diet. Animals were housed in humidity- and temperature-controlled conditions, with light/dark cycles set to 12-h intervals, and maintained under prescribed and opportunistic pathogen-free conditions. All animal studies were performed according to the experimental protocol approved by the Animal Care and Use Committee of Chang Gung University (Taipei, Taiwan).

acute liver animal model of failure

Mice were randomly divided into 4 groups. Test compounds (5 or 10 mg/Kg) or control solvent (80% saline, 10% ethanol, 10% Tween-20) were injected via the tail vein of each mouse. Mice were allowed to rest for 1 h before acute liver failure was induced by intraperitoneal injection of LPS (40 μg/ml)/D-GalN (400 mg/kg). Animals were sacrificed 5 hours later, and blood samples and tissue samples were collected separately. The blood samples were analyzed for respective levels of LDH, AST, ALT, BUN and CRE. Liver tissue was fixed with 10% formalin, sectioned, and stained with hematoxylin and eosin (H&E).

acute lung injury (ALI) animal model

ALI was induced by administering LPS (2 mg/kg) to 7-8 week old mice (C57BL/6) by intratracheal spray. After an overnight fast, the mice were injected intravenously with ribasitinib (10 mg/kg) or an equivalent amount of solvent (vehicle). One hour later, LPS (2 mg/kg in 40 μL 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, sectioned, and stained with hematoxylin and eosin (H&E).

Example 1 The compounds of the present invention inhibit the LPS active d-THP-1 nigericin-induced IL-1 beta secretion

In this example, the effect of a compound of the invention (ie, ribasitinib) on the release of IL-1β from LPS-activated macrophage-like cells (ie, d-THP-1 cells) was investigated. To do this, d-THP-1 cells were first activated with LPS (100 ng/ml) and then with various concentrations (ie 0.01, 0.03, 0.1, 0.3 or 1 μM) of the compounds of the invention or glyburide (50 μM) , positive control), and finally stimulated with stimulators (i.e., nigericin (5 μM), MSU (100 μg/mL), or IMQ (100 μM)), and the results are shown in Figures 1 to 3.

As shown, nigericin resulted in the release of significant amounts of IL-1β from LPS-activated d-THP-1 cells, which was reduced by the compounds of the present invention (Figure 1). Similarly, MSU and IMQ also resulted in a massive release of IL-1β, which was inhibited by the compounds of the present invention, respectively (Figures 2 and 3).

Example 2 The compounds of the present invention inhibit the LPS active d-THP-1 nigericin-induced IL-18 secretion

In this example, the effect of a compound of the present invention (ie, ribasitinib) on the release of IL-18 from LPS-activated macrophage-like cells (ie, d-THP-1 cells) was investigated. To do this, d-THP-1 cells were first activated with LPS (100 ng/ml) and then with various concentrations (ie 0.01, 0.03, 0.1, 0.3 or 1 μM) of the compounds of the invention or glyburide (50 μM) , positive control), and finally stimulated with nigericin (5 μM), the results are shown in Figure 4. As shown, nigericin resulted in the release of significant amounts of IL-18 from LPS-activated d-THP-1 cells, and this release was significantly reduced by the compounds of the invention (Figure 4).

Example 3 Cytotoxicity studies of the compounds of the present invention

In this example, the cytotoxic effects of the compounds of the present invention were investigated by measuring LDH. The results are shown in FIG. 5 .

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

Example 4 The compounds of the present invention inhibit the LPS active d-THP-1 nigericin-induced pyroptosis and pyroptosis in cells LDH release

Pyroptosis is a form of programmed cell death. During this process, immune cells recognize foreign danger signals within themselves, release pro-inflammatory cytokines, swell, burst, and then die. In this example, the effect of compounds of the present invention on nigericin-induced pyroptosis and LDH release was investigated, wherein pyroptosis was observed using field emission scanning electron microscopy and LDH levels were determined by enzymatic assays.

Reference is made to Figure 6, which is a photograph of LPS-activated d-THP-1 cells stimulated with nigericin in the presence or absence of compounds of the present invention. As expected, nigericin induced swelling and lysis of LPS-activated d-THP-1 cells, but pretreatment with compounds of the present invention and glyburide (positive control) significantly reduced pyroptosis . In addition, the compounds of the present invention were observed to inhibit the release of LDH (Figure 7).

Example 5 The compounds of the present invention inhibit the LPS active d-THP-1 nigericin-induced ASC spot formation

A hallmark of inflammasome activation is the formation of ASC puncta, which are micron-scale structures formed by the inflammasome adaptor protein ASC (CARD-containing apoptosis-associated puncta-like protein), consisting of a pyrin domain (PYD) and a sulfur It consists of a caspase recruitment domain (CARD). In the present example, the effects of the compounds of the present invention on nigericin-induced ASC spot formation in LPS-activated d-THP-1 cells were observed using a fluorescence microscope, and the results are shown in FIG. 8 .

It is clear from the photographs in Figure 8 that after treatment with nigericin, distinct ASC spots are formed, however, if the cells are pretreated with a compound of the invention or glibenclamide (positive control), the Reduces the formation of ASC spots. The data in this example clearly show that the compounds of the present invention inhibit the inflammasome from being activated.

Example 6 The compounds of the present invention reduce the LPS/D-GalN induced acute liver injury

In this example, an animal model of acute liver injury was used to study the effect of the compounds of the present invention on acute liver failure. Briefly, animals were first treated with LPS/D-GalN (LPS: 40 μg/mL; D-GalN: 400 mg/mL) to induce acute liver injury, then, before and after treatment with compounds of the invention, respectively , blood and tissue samples are taken, which are then analyzed biochemically and microscopically. The results are shown in Figures 9 and 10.

It is evident from Figure 9 that LPS/D-GalN treatment causes liver damage, as the overall image of the damaged liver tissue is dark red, a sign of tissue hemorrhage (Figure 9, panel A). In contrast, images taken from liver samples from animals treated with compounds of the present invention showed a marked reduction in bleeding and inflammation, which was also reflected in a reduction in tissue weight (Figure 9, panel B). In addition, various indicators of liver tissue damage, including ALT, AST, CRE and LDH, were significantly reduced in their respective levels after treatment with the compounds of the present invention. Example 7 Compounds of the present invention attenuate LPSN -induced acute lung injury (ALI) in mice

In this example, an animal model of ALI was used to study the effect of the compounds of the present invention on ALI. Briefly, 7-8 week old beetles were treated with an intratracheal spray of LPS (2 mg/Kg) to induce ALI. After one night of fasting, mice were injected intravenously with ribasitinib (10 mg/kg) or an equivalent amount of solvent, and 1 hour later, LPS (2 mg/kg, dissolved in 400 mg/kg) was intratracheally sprayed. μL of 0.9% saline) or 0.9% saline to induce ALI in animals. Mice were sacrificed after 5 hours, and their lung tissues were harvested. Tissues were fixed with 10% formalin and then stained with H&E. The results are shown in FIG. 11 .

The photograph from Figure 11 shows that ALI does not appear in the lung tissue treated with the solvent; on the contrary, the LPS-treated lung tissue has altered cell morphology and infiltration. As for the treatment of ribasitinib (10 mg/kg) before LPS-induced ALI, the degree of lung tissue morphological changes or infiltration caused by LPS were significantly improved. This result shows that ribasitinib can improve or cure ALI.

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

It will be understood that the above description of the embodiments is given by way of example only, and various modifications may be made by those skilled in the art to which the present invention pertains. 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 the present invention have been described above with a certain degree of characterization or with reference to one or more separate embodiments, those of ordinary skill in the art to which this invention pertains can understand all embodiments of the invention without departing from the scope of the invention. Various changes are made to the disclosed embodiments.

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The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fee.

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 present invention. The present description will be better understood from the following detailed description read with reference to the accompanying drawings, in which:

Figure 1 : Rebastinib inhibits nigericin-induced IL-1β secretion in lipopolysaccharide ( LPS ) -activated d -THP-1 cells . LPS-activated d-THP-1 cells were incubated in DMSO (0.1%, as control), ribasitinib (0.01-1 μM), or glibenclamide (50 μM) for 30 min, and then treated with Treat with nigericin (5 μM) or solvent for 30 min. The concentration of IL-1β in the cell supernatants was analyzed with an ELISA kit. Data are shown as mean ± SEM (n = 6). ** p < 0.01 compared to control.

Figure 2 : Ribasatinib inhibits monosodium urate (MSU ) -induced IL-1β secretion in LPS - activated d-THP-1 cells . LPS-activated d-THP-1 cells were incubated in DMSO (0.1%, as control), ribasitinib (0.003-1 μM) or glyburide (50 μM) for 30 min, and then treated with MSU (100 μM) for 30 min. μg/ml) or solvent treatment for 4 hours. The concentration of IL-1β in the cell supernatants was analyzed with an ELISA kit. Data are shown as mean ± SEM (n = 6). * p <0.05; ** p <0.01; *** p < 0.001 compared to control.

Figure 3 : Ribasitinib 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 control), ribasitinib (0.003-1 μM), or glibenclamide (50 μM) for 30 min, and then treated with IMQ ( 100 μM) or solvent treatment for 2 hours. The concentration of IL-1β in the cell supernatants was analyzed with an ELISA kit. Data are shown as mean ± SEM (n = 6). ** p <0.01; *** p < 0.001 compared to control.

Figure 4 : Ribasatinib inhibits nigericin-induced IL-18 secretion in LPS - activated d-THP-1 cells . LPS-activated d-THP-1 cells were incubated in DMSO (0.1%, as control), ribasitinib (0.01-1 μM), or glyburide (50 μM) for 30 min, and then treated with Nigeria prime (5 μM) or solvent for 30 min. The concentration of IL-18 in the cell supernatants was analyzed with an ELISA kit. Data are shown as mean ± SEM (n = 6). * p < 0.05 compared to control.

Figure 5 : Ribasitinib does not alter cell viability of LPS - activated d-THP-1 cells. LPS-activated d-THP-1 cells were cultured in DMSO (0.1%, as control), ribasitinib (0.01-1 μM), glibenclamide (50 μM), or Triton X-100 (0.1%, for total lactate dehydrogenase (LDH) release) for 1 hour. The concentration of LDH in the cell supernatants was analyzed by the CytoTox 96 non-radioactive cytotoxicity assay. Data are shown as mean ± SEM (n = 6).

Figure 6 : Ribasitinib inhibits nigericin-induced pyroptotic cell death in LPS - activated d-THP-1 cells . d-THP-1 cells activated by LPS were incubated in DMSO (0.1%), ribasitinib (1 μM) or glibenclamide (50 μM) for 30 min, and then treated with nigericin (5 μM) for 30 min. ) or solvent treatment for 30 minutes. Representative images of pyroptosis were examined by scanning electron microscopy (n = 4).

Figure 7 : Ribasatinib inhibits nigericin-induced pyroptotic LDH release in LPS - activated d-THP-1 cells. LPS-activated d-THP-1 cells were incubated in DMSO (0.1%, as a control), ribasitinib (0.01-1 μM), glibenclamide (50 μM), or Triton X-100 for 30 min, It was then treated with nigericin (5 μM) 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 ± SEM (n = 6). ** p &lt;0.01; *** p &lt; 0.001 compared to control.

Figure 8 : Ribasatinib inhibits nigericin-induced ASC puncta formation in LPS - activated d-THP-1 cells . LPS-activated d-THP-1 cells were incubated in DMSO (0.1%, as control), ribasitinib (1 μM), or glibenclamide (50 μM) for 30 min, and then treated with nigericin ( 5 μM) or solvent treatment for 30 min. Cells were fixed with 4% paraformaldehyde and stained with anti-ASC and hoeschst33342 (n = 2). A fluorescence microscope (IX81, Olympus) was used to inspect and obtain fluorescence pictures.

Figure 9 : Ribasitinib attenuates LPS/ D -galactosamine (D-GalN) -induced acute liver injury in mice. The vehicle or ribasitinib (5 or 10 mg/kg) was administered intravenously to mice (C57BL/6) at 7-10 weeks of age for approximately 1 hour, followed by an intraperitoneal injection of LPS. (40 μg/kg)/D-GalN (400 mg/kg) was administered to mice for about 5 hours to induce acute liver injury. (A) Representative gross images of liver morphology were taken with a digital camera (scale bar: 1 cm). (B) Whole liver weights are expressed as mean ± SEM (n = 2-4). * p <0.05; compared to LPS/D-GalN treatment.

Figure 10 : Ribasitinib attenuates serum biochemical parameters in LPS/D-GalN -induced mice. Mice (C57BL/6) at 7-10 weeks of age were treated with vehicle or ribasitinib (5 or 10 mg/kg) by intravenous injection, followed by LPS (40 μg/kg)/D-GalN ( 400 mg/kg) by intraperitoneal injection for an additional 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 lines are expressed as mean ± SEM (n = 2-4). * p <0.05; ** p < 0.01 compared to LPS/D-GalN treatment alone.

Figure 11 : Ribasitinib attenuates LPS -induced acute lung injury. 7-8 week old mice (C57BL/6) were treated with vehicle or ribasitinib (10 mg/kg) by intravenous injection, and 1 hour later, LPS (2 mg) was administered by intratracheal spray. /kg, dissolved in 40 μL of 0.9% normal saline). After 5 hours, the mice were sacrificed and their lung tissues were collected and stained with hematoxylin and eosin (H&E).

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Claims (3)

A kind of 4-[4-[5-tert-butyl-2-quinoline-6-ylpyrazol-3-yl]-amine carboxylamino]-3-fluorophenoxy]-N-methyl Use of pyridine-2-carboxamide, its salts, solvates or esters for the preparation of medicaments for the treatment of acute liver failure or acute lung injury (ALI). The use of claim 1, wherein the ALI is transfusion-related lung injury, ventilator-induced klung injury, bacteria-induced lung injury, or virus-induced lung injury. The use of claim 1, wherein the individual is a mammal.

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