TW202241392A - Methods for the treatment of diseases associated with dysregulated activation and recruitment of neutrophils - Google Patents

Abstract

Disclosed herein is a novel use of 3,3'-dihydroxy-2',6'-bis(p-hydroxybenzyl)-5-methoxybibenzyl (or bletinib), a salt, a solvate or an ester thereof for manufacturing a medicament suitable for the treatment of diseases and/or disorders associated with the dysregulated activation and recruitment of neutrophils, such as acute lung injury (ALI).

Description

Methods for treating diseases associated with dysregulation of neutrophil activation and chemotaxis

The present invention relates to a new use of natural bibenzyl, and in particular to 3,3'-dihydroxy-2',6'-bis(p-hydroxybenzyl)-5-methoxydibenzyl (or It is called britinib (bletinib)) for the treatment of diseases and/or disorders (such as acute lung injury) related to neutrophil activation disorder and chemotaxis.

Neutrophils are the most abundant granular white blood cells in the systemic circulation. They are responsible for eliminating pathogens through degranulation, releasing neutrophil elastase (NE) and exacerbating the amount of superoxide in the respiratory tract. Increase and form the neutrophil extracellular trap (NET). Thus, neutrophils are key effectors of the acquired and innate immune systems. During inflammation, key steps in neutrophil chemotaxis include adhesion and migration, which are regulated by macrophage-1 antigen (Mac-1; also known as αMβ2 and CD11b-CD18) conformational changes on the neutrophil surface Regulated. Dysregulation of neutrophil activation and chemotaxis can cause damage to host tissues through the release of excessive proteolytic enzymes, reactive oxygen species (ROS) and neutrophil extracellular bactericidal network, resulting in various Diseases, including autoimmune diseases (eg, systemic lupus erythematosus, rheumatoid arthritis, and psoriasis), infectious diseases (eg, sepsis), inflammatory diseases (eg, acute respiratory distress syndrome, chronic obstructive pulmonary disease, and asthma) , atherosclerosis and other major diseases such as cancer.

Traditional Chinese medicine has used Bletilla tuber thousands of years ago to treat inflammatory and bleeding disorders of the lungs, gastrointestinal tract and skin. Brittatinib (3,3'-dihydroxy-2',6'-bis(p-hydroxybenzyl)-5-methoxydibenzyl) is a natural bibenzyl, which was first extracted from the bulb of Bletilla striata have antibacterial, antifungal, antiallergic and antimitotic activities.

In the present application, the inventors unexpectedly found that britinib can regulate the inflammation of activated human neutrophils. Therefore, britinib can be used as a therapeutic agent for the treatment of neutrophil activation disorders and chemotaxis-related neutrophils. Candidate compounds for drugs of diseases and/or disorders (eg, acute lung injury).

The invention provides a new application of natural bibenzyl, which is 3,3'-dihydroxy-2',6'-bis(p-hydroxybenzyl)-5-methoxydibenzyl extracted from Bletilla striata (or known as britinib), unexpectedly found that it can inhibit neutrophil activation disorders, so britinib can be used as a treatment for neutrophil activation disorders and chemotaxis-related diseases and/or disorders (for example, acute Candidate compounds for drugs against lung injury).

Therefore, the first aspect of the present invention relates to the use of britinib, its salt, solvate or ester in the preparation of a drug suitable for treating acute lung injury. The method comprises administering to the patient an effective amount of britinib, a salt, solvate or ester thereof.

According to an embodiment of the present invention, the acute lung injury may be acute respiratory distress syndrome (acute respiratory distress syndrome, ARDS).

Examples of acute respiratory distress syndrome treatable by the method of the present invention include, but are not limited to, transfusion-related lung injury, ventilator-induced lung injury, bacterial-induced lung injury, virus-induced lung injury, and the like.

According to an embodiment of the present invention, Brittatinib is present in the drug in an amount ranging from 0.1 mg to 60 g. Preferably, Brittatinib is present in the medicament in an amount of 2 mg to 5 g.

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

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 claims.

After referring to the following embodiments, those with ordinary knowledge in the technical field of the present invention can easily understand the basic spirit and other invention objectives of the present invention, as well as the technical means and implementation modes adopted by the present invention.

In order to make the description of the present disclosure more detailed and complete, the following provides an illustrative description of the implementation aspects and specific embodiments of the present invention; but this is not the only form of implementing or using the specific embodiments of the present invention. The description covers features of various embodiments as well as method steps and their sequences for constructing and operating those embodiments. However, other embodiments can also be used to achieve the same or equivalent functions and step sequences.

Unless otherwise defined in this specification, the meanings of scientific and technical terms used herein are the same as those commonly understood and commonly used by those skilled in the art to which this invention belongs.

Where there is no conflict with the context, the singular nouns used in this specification include the plural forms of the nouns; and the plural nouns used also include the singular forms of the nouns. In addition, in this specification and the scope of the patent application, expressions such as "at least one" and "one or more" have the same meaning, and both of them mean that one, two, three or more are included. What's more, in this specification and the scope of patent application, "at least one of A, B and C", "at least one of A, B or C" and "at least one of A, B and/or C ” means to cover only A, only B, only C, both A and B, both B and C, both and C, and all three of A, B and C.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the relative numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently inherently contain standard deviations resulting from their individual testing methodology. Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specified value or range. Alternatively, the term "about" means that the actual value falls within acceptable standard error of the mean, as considered by one of ordinary skill in the art to which this invention pertains. Except for the experimental examples, or unless otherwise expressly stated, all ranges, quantities, numerical values and percentages used herein should be understood to be used (for example, to describe the amount of material used, the length of time, temperature, operating conditions, quantitative ratios and other similar Those) are modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the appended patent claims are approximate values and may be changed as required. At least these numerical parameters should be understood as the value obtained by applying the normal rounding method to the indicated effective digits. Herein, numerical ranges are expressed as being from one endpoint to another point or between two endpoints; unless otherwise stated, the numerical ranges stated herein are inclusive of the endpoints.

1. Glossary

The term "salt" as used herein means a pharmaceutically acceptable salt which, within sound medical judgment, is suitable for contact with tissues of humans and lower animals without undue toxicity, irritation, allergic reaction, etc. , and commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are amino acids with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, acid, succinic acid, or malonic acid), or 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, cyclopentanepropionate, digluconate, lauryl sulfate, ethanesulfonate, formate, fumarate, glucosulphate, glucose Sugar heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, Malonate, Methanesulfonate, 2-Naphthalenesulfonate, Nicotinate, Nitrate, Oleate, Oxalate, Palmitate, Palmitate, Pectate, Persulfate Phenylpropane salt, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate , valerate, etc. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (C _1-4 alkyl) ⁴ -salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts, including non-toxic ammonium, quaternary ammonium and amine cations where appropriate with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkanes Salts formed by base sulfonate ions and aryl sulfonate ions.

The term "solvate" as used herein refers to a form of a compound that is associated with a solvent, usually by a solvolytic 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 can be prepared, eg, in crystalline form, and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some cases, solvates will be able to be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. "Solvate" includes both solution-phase and isolatable solvates. Representative solvates include hydrates, ethanolates and methanolates.

The terms "administered", "administering or administration" are used interchangeably herein to refer to modes of delivery including, but not limited to, intravenous, intramuscular, intraperitoneal, intraarterial, intracranial or The agent (eg, a compound or composition of the invention) is administered subcutaneously. In some embodiments, a compound of the invention, or a salt, solvate thereof, is formulated as a lozenge for oral administration. In other embodiments, the compound of the present invention or a salt thereof, or a solvate thereof is formulated into a powder, and mixed with a suitable carrier (such as a buffer) before use, such as intravenous injection.

An "effective amount" of a compound described herein (administered alone or in combination with another agent) is an amount sufficient to elicit a desired biological response, such as inhibiting the activation of inflammation or reducing the target disease or disease-associated disease described herein. associated symptoms. As will be understood by those of ordinary skill in the art, effective amounts of the compounds described herein may vary according to factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the patient. In some instances, an effective amount may be a therapeutically effective amount, which refers to a therapeutic dose sufficient, alone or in combination with other therapies, to provide a therapeutic benefit or delay onset in the treatment of a disorder, or to minimize one or more of the disorders associated with the disorder. symptom. A therapeutically effective amount is an amount that improves overall treatment, reduces or avoids symptoms, signs or causes of a disorder, and/or enhances the therapeutic efficacy of another therapeutic agent. In other instances, the effective amount may be a prophylactically effective amount. A prophylactically effective amount of a compound refers to a therapeutic dose, alone or in combination with other agents, which provides a prophylactic benefit in preventing the condition. For example, a prophylactically effective amount of a compound may be an amount sufficient to prevent or delay the onset of the disorder, or prevent or delay the onset of one or more symptoms associated with the disorder, or prevent recurrence of the disorder or one or more associated symptoms. It can also be an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

2. Uses of the compounds of the invention

The present invention unexpectedly discovered natural 3,3'-dihydroxy-2',6'-bis(p-hydroxybenzyl)-5-methoxydibenzyl (or Brittany) extracted from Bletilla striata striata It can treat activated neutrophil disorders in humans. Therefore, britinib can be used as a candidate compound for the development of drugs suitable for the treatment of diseases or conditions related to neutrophil activation dysregulation and chemotaxis (eg, acute lung injury (ALI) and the like).

Therefore, the first aspect of the present invention is to provide the use of britinib, its salt, solvate or ester in the preparation of a drug suitable for the treatment of ALI.

The compounds of the present invention can be purchased from commercial sources, or isolated by any method known in the art, such as the method previously described by Lin et al. (J. Nat. Prod. 2016, 79, 1911-1921). Bioactivity assays of this compound revealed that it is a potent inhibitor of superoxide anion production, reactive oxygen species (ROS) production and degranulation in activated human neutrophils. In addition, britinib had no cytotoxicity or superoxide-scavenging activity in a xanthine/xanthine oxidase cell-free system. The present invention demonstrates that britinib can be used as a candidate compound for the development of drugs suitable for the treatment of diseases associated with dysregulation of neutrophil activation and chemotaxis (eg, ALI).

Examples of acute lung injury that may be treated with compounds of the invention include, but are not limited to, acute respiratory distress syndrome (ARDS), which may be transfusion-related lung injury, ventilator-induced lung injury, bacterial-induced lung injury , virus-induced lung injury, etc.

According to an embodiment of the present invention, the compound of the present invention, namely 3,3'-dihydroxy-2',6'-bis(p-hydroxybenzyl)-5-methoxybibenzyl (or Brittany) , is administered to a patient in an amount of 0.01 to 100 mg/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, 0.03, 0.04, 0.05, 0.09, 0.1, 0.2, 0.3, 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, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 100 mg /kg; Preferably, 3,3'-dihydroxy-2',6'-bis(p-hydroxybenzyl)-5-methoxydibenzyl (or Brittatinib) is 0.1 to 80 mg/kg The amount in kilograms is administered to the patient, 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, 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, Brittatinib is administered to the patient in an amount of 2 mg/kg. An effective amount of a 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 together with suitable carriers or excipients for a suitable route of administration, such as oral, parenteral, spray by inhalation, topical, rectal, nasal, buccal, vaginal or implanted. Inserted reservoir. The term "injection" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. Preferably, the britinib is present in the preparation in an amount of about 0.1 mg to about 60 g; such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 , 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450 , 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 390, 700 , 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950 , 960, 970, 980, 990 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 grams. More preferably, said britinib is present in the formulation in an amount from about 2 mg to about 5 g, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 mg, 1, 2, 3, 4 and 5 gram.

Sterile injectable formulations, such as sterile injectable aqueous or oily suspensions, can be formulated according to techniques known in the art with suitable dispersing or wetting agents (such as TWEEN® 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a diluent or solvent that is non-toxic and injectable, for example, a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be employed include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium (for example, 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, are commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms which may also be used for formulation purposes.

Formulations suitable for oral administration may be in any orally acceptable dosage form including, but not limited to, capsules, lozenges, emulsions and aqueous suspensions, dispersions and solutions. In the case of oral lozenges, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also usually added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions or emulsions are administered orally, the compounds of the invention can be suspended or dissolved in an oily phase in combination with emulsifying or suspending agents. Certain sweetening, flavoring or coloring agents may be added, if desired. Nasal aerosol or inhalation formulations may be prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as a solution in saline, benzyl alcohol, or with other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons and/or Or other solubilizers or dispersants known in the art. The compounds of this 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 emulsifying agents, disintegrating agents , adhesives, 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.

The excipients present in the formulations of the invention must be "pharmaceutically acceptable", i.e. the excipients are compatible with the active ingredients of the pharmaceutical composition (and preferably, are capable of stabilizing the pharmaceutical composition) and are The patient is harmless. For example, solubilizers such as cyclodextrins, which form specific, more soluble complexes with the compounds of the invention, can serve as pharmaceutically acceptable excipients for delivering the compounds of the invention to patients. Examples of other pharmaceutically acceptable excipients include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&C Yellow #10.

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

In certain embodiments, a kit as described herein is used to inhibit neutrophil activation and chemotaxis dysregulation. In certain embodiments, a kit as described herein is used in a patient in need thereof to treat any target disease as described herein (eg, ALI). Accordingly, any kit described herein may include instructions for the administration of the compounds or pharmaceutical compositions contained therein. The kits of the invention may also include information required by regulatory agencies such as the FDA. In certain embodiments, kits and instructions are provided for treating the diseases described herein. In certain embodiments, kits and instructions are provided for preventing the diseases described herein. The kits of the invention may include one or more of the additional agents described herein as a single composition.

A "patient" suitable for treatment by the methods described herein can be a human patient (such as a pediatric patient, e.g., an infant, child, or adolescent, or an adult patient, e.g., a young, middle-aged, geriatric, or geriatric patient), or a non-human animal, e.g. : Dogs, cats, cows, pigs, horses, sheep, goats, rodents (eg mice, rats) and non-human primates (eg cynomolgus monkeys, rhesus monkeys). A non-human mammal can be a transgenic or genetically engineered animal. In some examples, the patient is a human patient who has, is suspected of having, or is at risk of having a disease of interest described herein (ie, ARDS). In some embodiments, the patient is a human or non-human mammal suspected of having a disorder secondary to neutrophil activation and dysregulation of chemotaxis (eg, ARDS).

It is also understood that a compound or formulation as described herein may be used in combination with one or more additional pharmaceutical agents (eg, therapeutically and/or prophylactically active agents) in any of the methods described herein. Compounds or agents can be used in combination with additional agents, administered to a patient in need thereof, to enhance activity (eg, activity (eg, potency and/or efficacy) to treat, prevent the diseases described herein, and inhibit neutrophils in patients It is also understood that the therapy employed may achieve the desired effect for the same condition, and/or it may achieve a different effect.

The present invention will now be described more specifically with reference to the following examples, which are provided by way of illustration and not limitation. While they can generally be used, other procedures, methods or techniques known to those skilled in the art can be used instead.

Example

Materials and methods

Preparation of Brittatinib

Brittatinib was extracted and purified according to the method previously described by Lin et al. (Lin et al., J. Nat. Prod. 2016 79, 1911-1921). Briefly, britinib was extracted from the rhizome of sea-island ramie ( B. formosana ) with ethanol at 60 °C and purified by column chromatography. Brittatinib (purity > 98%) was dissolved in dimethyl sulfide (DMSO) as the compound mother solution. The DMSO control concentration used in cell experiments was 0.1% and did not affect the measured parameters.

animal

Animal care and experimental protocols were approved by the Animal Care and Use Committee of Chang Gung University, Taiwan. Furthermore, animal studies were performed according to ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. All experimental procedures were in accordance with "Guide for the Care and Use of Laboratory Animals" ( The Guide for the Care and Use of Laboratory Animals ) (National Research Council Update Laboratory Care and Use Guide/National Research Council Committee for the Update of the Guide for the Care and Use of Laboratory, 2011). 8-week-old Specified pathogen-free (SPF) BALB/c male mice (weight: 20±1 g) were purchased from BioLASCO (Taiwan). Five mice shared a ventilated cage with standard bedding and had free access to water and food. All mice were housed in the SPF animal facility with a 12-12 hr light-dark cycle. Mice were acclimatized for at least 1 week before being used in experiments.

LPS induced ALI and mortality model

A total of 24 BALB/c male mice were randomly divided into four groups (6 mice/group) and administered: vehicle, britinib, LPS, britinib+LPS. Mice were fasted overnight, and then injected intraperitoneally with 50 μL of Brittatinib (25 mg/kg) or 50 μL of vehicle (10% DMSO). Mice were generally anesthetized with xylazine (6 mg/kg) and Zoletil 50 (30 mg/kg), and 50 μL of LPS (from E. coli O111:B4; 2 mg/kg) or 50 μL of 0.9 % saline (in vehicle group and britinib group), to induce ALI in mice. Five hours later, after the mice were sacrificed, the lung tissues of the mice were collected and frozen to measure the activity of myeloperoxidase (MPO); or the tissues were fixed with 10% formalin for tissue sectioning and Immunofluorescence staining.

For the LPS-induced mortality model, mice were injected intraperitoneally with a single 50-μL dose of LPS (from E. coli O111:B4; 5 mg/kg) or 0.9% saline (vehicle alone group). Mice were monitored for 5 days to determine survival.

Histological Sections and Immunofluorescent Staining

Mouse lung tissues were washed with phosphate-buffered saline (PBS) and fixed in 10% formalin for 24 hours. Samples were then dehydrated, embedded in paraffin, microtome-sectioned into 3 μm thick sections, and mounted on glass slides. The sections were stained with hematoxylin and eosin (H&E) and the corresponding antibodies. Then, images were captured with an optical microscope.

For immunofluorescent staining, sectioned tissue was mixed with anti-H3 (citH3; citrulline R2+R8+R17, diluted 1:800) and anti-Ly6G antibodies (diluted 1:200) cultivated afterwards. Anti-citH3 labeled with Alexa Fluor 488 or anti-Ly6G anti-IgG secondary antibodies labeled with Alexa Fluor 568 were used after dilution of 1:1000 and 1:500, respectively. Immunofluorescence images were captured with a confocal microscope (LSM 510 Meta, Zeiss).

Determination of myeloperoxidase (MPO) activity

Mouse lung tissue was suspended in 0.5% cetyltrimethylammonium bromide buffer (pH 6.0) and homogenized by sonication. To assess MPO activity, MPO substrate buffer (containing PBS, 0.0005% hydrogen peroxide and 0.2 mg/mL O-dianisidine hydrochloride) was added to the homogenized tissue, and the absorbance at 460 nm was detected by a spectrometer , and then, the MPO activity was calculated with reference to the human MPO activity standard curve.

Isolation of human neutrophils

This study was conducted after approval by the Institutional Review Board of Chang Gung Memorial Hospital (IRB No. 201601111A3) in accordance with the Declaration of Helsinki. After obtaining the written informed consent of the subjects, blood samples were drawn from 20-30-year-old healthy individuals who had not taken any drugs in the past 2 weeks. Neutrophils were then isolated using standard procedures including Ficoll-Hypaque gradient centrifugation, dextran sedimentation, and hypotonic erythrocyte lysis. The isolated neutrophils were then suspended in Ca2 ⁺ -free HBSS (pH 7.4) and stored at 4°C until use.

Measure the amount of extracellular superoxide anion

The amount of extracellular superoxide anion produced by activated neutrophils was assessed by the reduction of sidericytochrome c. Incubate neutrophils (6×10 ⁵ cells/mL) with Ca ²⁺ (1 mM) and siderocytochrome c (0.5 mg/mL) at 37 °C, and then incubate with 0.1% DMSO or 0.3–10 µM Brittatinib was incubated for 5 min. Cells were pretreated with cytochalasin B (CB, 1 or 2 μg/mL) for 3 minutes and then stimulated with fMLF, MMK-1 or sodium fluoride (NaF), or directly with phorbol-12 -Myristate-13-acetate (phorbol-12-myristate-13-acetate, PMA) activates cells. Changes in absorbance at 550 nm were continuously detected using a spectrometer (U-3010, Hitachi, Tokyo, Japan), and superoxide was calculated using a previously described method (Hwang et al., 2003 Mol. Pharmacol. 64(6), 1419-1427). Anion content.

Measuring the amount of superoxide anion in cells

Human neutrophils (2.5 × 10 ⁶ cells/mL) were labeled with 2 μM dihydrorhodamine 123 (DHR123) for 10 min at 37°C, then incubated with DMSO or britinib for 5 min, followed by 0.1 Stimulate with μM fMLF/0.5 μg/mL CB for 15 minutes. Fluorescence intensity was detected by flow cytometry to evaluate the intracellular superoxide anion content of human neutrophils.

Measuring the amount of total ROS

In a 96-well plate, human neutrophils (2 × 10 ⁶ cells/mL) were pre-incubated with 6 U mL ^-1 horseradish peroxidase (HRP) and 37.5 μM luminol at 37°C. Incubate for 5 minutes. Cells were incubated with DMSO or Brittatinib for 5 minutes and then stimulated with 0.1 μM fMLF. Chemiluminescence was then detected and analyzed instantaneously on a 96-well plate chemiluminescence meter (Tecan Infinite F200 Pro; Männedorf, Switzerland).

Measure the release of NE

After treatment with 1 mM CaCl ₂ and 100 μM NE substrate (methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide, Methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide), Human neutrophils (6 × 10 ⁵ cells/mL) were incubated with DMSO or Brittatinib at 37°C for 5 minutes. Stimulate cells with fMLF/0.5 μg mL-1 CB, leukotriene B4 (LTB4)/2 μg mL ^-1 CB, NaF/2 μg mL ^-1 CB or MMK-1/0.5 μg mL ^-1 CB10 Minutes, and then measure the change of absorbance at 405 nm with a spectrometer to measure the amount of released NE.

Measurement of production of neutrophil extracellular trap (NET)

Quantification of the amount of extracellular DNA

Human neutrophils (10 ⁶ cells/mL) resuspended in HBSS containing 2.5 μM Sytox green were incubated with DMSO or britinib for 10 minutes and stimulated with 10 nM PMA or 10 μg/mL LPS3 Hour. Fluorescence intensity at wavelengths between 485–535 nm was measured with a Tecan Infinite 200 analyzer.

Capture NET photos

Neutrophils (3 × 10 ⁵ cells/mL) were incubated with DMSO or britinib for 10 minutes and then activated with 10 nM PMA for 2 hours. Neutrophils were fixed with 4% paraformaldehyde, treated with 5% goat serum blocker for 1 hour, and then treated with 5 μg/mL anti-MPO antibody (Abcam) and 5 μg/mL anti-NE antibody (Merck Millipore ) for 1 hour. The cells were then treated with Alexa 488 or 568-labeled goat anti-rabbit secondary antibody for an additional 1 hour. Thereafter, cells were washed with PBS and treated with 1 ng/mL Hoechst 33342 and ProLong Gold antifade reagent (Invitrogen, CA, USA). NETs generated in activated neutrophils were observed by immunofluorescence microscopy and scanning electron microscopy, respectively.

Assess the adhesion of neutrophils

Human neutrophils (10 ⁶ cells/mL) were labeled with Hoechst 33342, and then incubated with DMSO or britinib for 5 minutes. After centrifugation, cells were resuspended and activated with 0.1 μM fMLF/1 μg/mL CB for 10 minutes, then co-incubated with bEnd.3 cells for 30 minutes at 37°C. After washing with HBSS, cells were fixed with 4% paraformaldehyde, and the number of neutrophils adhered to bEnd.3 cells was observed and quantified on a motorized inverted microscope (Olympus, Japan).

Assess the migration of neutrophils

Chemotactic migration of neutrophils was assessed in a microchemotactic chamber with a 3-μm filter (Millipore). Neutrophils (5 × 10 ⁶ cells/mL) treated with britinib or DMSO for 5 minutes were placed in the top chamber of the microchemotactic chamber, and 0.1 μM fMLF was added to the bottom chamber. After incubation for 1 hour under 5% CO ₂ , the number of neutrophils migrating from the top chamber to the bottom chamber was counted with a MoxiZ automated cell counter (ORFLO).

Assess surface CD18 and CD11b expression

Neutrophils (5 × 10 ⁵ cells/mL) were first incubated with Brittatinib or DMSO for 5 minutes and then incubated with 0.1 μM fMLF/1 μg/mL CB for 5 minutes to activate neutrophils. After centrifugation at 200 g for 8 min at 4°C, cells were resuspended in 5% bovine serum albumin with FITC-conjugated anti-CD18 or anti-CD11b antibody and kept on ice for 15 min in the dark. Fluorescence intensity was then analyzed by flow cytometry.

Immunoblotting Analysis of Neutrophil Lysates

Neutrophils were incubated with Brittatinib or DMSO for 5 minutes at 37°C and activated with 0.1 μM fMLF for 30 seconds. Proteins were separated from neutrophil lysates by electrophoresis (12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to nitrocellulose membranes. Use immunoblotting to identify target proteins, using specific anti-p38, anti-p-p38, anti-Akt, anti-p-Akt S473, anti-ERK, anti-p-ERK, anti-JNK, anti- p-JNK, anti-Src, anti-p-SFKs Y416, anti-p-Src Y416, anti-Lyn, anti-p-Lyn (Y396), anti-Fgr, anti-p-Fgr (Y412), anti- Antibodies against Hck, anti-p-Hck (Y410), anti-Btk, anti-p-Btk Y223, anti-Vav or anti-p-Vav (Y174), and secondary anti-rabbit antibodies conjugated to HRP (Cell Signaling Technology). Signal intensity was detected and quantified using a UVP BioSpectrum imaging system (Analytik Jena, USA).

Assessing SFKs enzymatic activity

The kinase activity of SFK was assessed with the ADP-Glo Kinase Assay Kit (Promega, Fitchburg, USA) according to the manufacturer's instructions. Briefly, SFKs (Src, Lyn, Fgr or Hck), their substrates - 125 μM ATP and 1-10 μM britinib or 0.1-3 μM PP2 are added to the reaction buffer for 1 hr to initiate Kinase reaction. ADP-Glo reagent is used to terminate the kinase reaction and remove residual ATP; next, a kinase assay reagent that converts ADP to ATP is added and incubated for 30 minutes. Luciferin/luciferase luminescence was measured on an Infinite 200 Pro (Tecan, Switzerland).

Statistics and Analysis

All data are presented as box plots (median, min-max). All experiments were performed with one-way analysis of variance and Dunnett's multiple comparison test. Mice survival was analyzed using the log-rank (Mantel-Cox) test. All statistical calculations were performed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Differences with a p -value < 0.05 were considered statistically significant.

A number of experimental examples are provided below to illustrate certain aspects of the present invention, so as to facilitate those skilled in the art to implement the present invention, and these experimental examples should not be considered as limiting the scope of the present invention. It is believed that one skilled in the art can, after reading the description presented herein, fully utilize and practice the present invention without undue interpretation. All publications cited here are considered as a part of this specification in their entirety.

Experimental example 1 In vitro properties of Brittatinib

1.1 Brittatinib improves superoxide anion and ROS generation

In this example, the regulatory effect of britinib on the inflammatory response was explored by monitoring the amount of superoxide anion generated in human neutrophils activated by chemical inducers. Chemoattractants include formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF), NaF (a G protein-activated agent), MMK-1 (a selective type II formyl peptide receptor 2 (FPR2) agonist), and phorbol 12-myristate 13-acetate (phorbol 12-myristate 13-acetate, PMA). The result is shown in Figure 1.

According to the results in Figure 1, it can be found that britinib can improve the superoxide anion produced by fMLF-activated human neutrophils, and this improvement is positively correlated with the dose of britinib (IC ₅₀ = 0.62 ± 0.15 μM ; Figure 1, (A)). Similarly, britinib attenuated the extent of superoxide anion release from neutrophils activated by other chemoattractants, including MMK-1, sodium fluoride (NaF), and PMA (Fig. 1, (B), (C) and (D)). Furthermore, Brittatinib also did not exhibit any cytotoxicity (Fig. 1, (F)) or ROS scavenging activity (Fig. 1, (G)) in the cell-free xanthine and xanthine oxidase system.

Furthermore, whether britinib affects ROS generation in activated neutrophils was determined by flow cytometry and chemiluminescence assays. According to the quantitative results of flow cytometry analysis and luminol amplified chemiluminescence assay, britinib significantly inhibited the generation of intracellular ROS in fMLF-activated neutrophils, and the degree of inhibition was similar to the dose of britinib There was a positive correlation (Figure 2, (A) and (B)).

1.2 Brittatinib inhibits degranulation of activated human neutrophils

In this example, the effect of britinib on degranulation was studied by measuring the release of neutrophil elastase (NE) from activated neutrophils, since degranulation is a An important function of white blood cells during inflammation.

The results showed that britinib inhibited NE release in fMLF-activated human neutrophils (IC50 = 0.53 ± 0.07 μM), but not in resting neutrophils (Fig. 3, (A )). In addition, britinib could down-regulate the release of NE from neutrophils activated by MMK-1, NaF and LTB4, respectively, and this down-regulation was positively correlated with the concentration of britinib (Fig. 3, (B), ( C) and (D)).

1.3 Brittatinib attenuates the extracellular network of neutrophils (NET) Formation

Consisting primarily of granulins, proteases, and histone-coated chromatin filaments, NETs are critical in inflammatory and autoimmune diseases. To study the effect of britinib on NET formation, neutrophils were stained with Sytox green after activation with PMA (10 nM) and LPS (10 μg/mL).

Fluorescence spectroscopy results showed that britinib significantly reduced the amount of NET formation induced by PMA (Figure 4, (A)). In addition, immunofluorescent staining revealed that neutrophils co-stained with Hoechst 33342 and anti-MPO antibody and anti-NE antibody were found in NET (Fig. 4, (B)). Immunofluorescence staining and scanning electron microscopy images showed that britinib effectively inhibited NET formation (Figure 4, (B) and (C)).

1.4 Brittatinib inhibition f in activated neutrophils ERK with Src family kinase (SFK) Phosphorylation

SFKs and the MAPK/ERK pathway are known to play key roles in neutrophil degranulation, respiratory burst, NET formation and migration. Therefore, this example examines the effect of britinib on the phosphorylation of SFK, Akt, ERK, JNK, and p38 in neutrophils.

Immunoblotting results showed that in neutrophils activated by fMLF, SFKs, Akt (S473), ERK, JNK, p38, Src (Y416), Lyn (Y396), Fgr (Y412), Hck (Y410), Btk (Y223) and Vav (Y174) phosphorylation was increased. However, britinib significantly inhibited the phosphorylation of ERK, SFKs, Src (Y416), Lyn (Y396), Fgr (Y412), Hck (Y410), Btk (Y223) and Vav (Y174), but not Inhibits the phosphorylation of Akt, JNK, and p38 (Figure 5, 6A to 6F).

1.5 Brittatinib inhibition SFK active

SFK is a general term for non-receptor tyrosine kinases present in neutrophils, and the main expressed proteins are Src, Fgr, Hck and Lyn. SFKs are responsible for creating an inflammatory environment in the body. Cell-free ADP-Glo kinase assay confirmed that both britinib and PP2 (a specific inhibitor of SFK) inhibited the kinase activities of Src, Fgr, Hck and Lyn to the same degree as the concentration of britinib or PP2 are positively correlated with each other (Fig. 6G to 6J).

1.6 Brittatinib reduces adhesion and migration of activated human neutrophils

Both adhesion and migration are key steps in a cascade of neutrophil expulsion responses during inflammation. In this example, in order to confirm whether britinib interferes with the adhesion and migration process, human neutrophils (10 ⁶ cells/mL) were first labeled with Hoechst 33342, and then treated with britinib (1-10 μM) After preconditioning and stimulation with fMLF, activated human neutrophils were incubated with bEnd.3 cells for 30 minutes at 37°C. The neutrophils attached to the bEnd.3 cells were detected and counted using a fluorescent microscope. Quantitative results showed that britinib could inhibit the adhesion function of fMLF-activated neutrophils (Fig. 7, (A)). Furthermore, using a chemotaxis chamber and a cell counter to count the number of neutrophils migrating, it was found that britinib significantly reduced fMLF-induced neutrophil migration (Fig. 7, (B)). In addition, IL-8 is a chemokine that can attract neutrophils, and it was observed that IL-8-induced neutrophil migration was also inhibited by britinib (data not shown).

1.7 Brittatinib reduces activated neutrophils Mac-1 Performance

Mac-1 is a complement receptor consisting of CD11b (integrin α _M ) and CD18 (integrin β ₂ ) that promotes leukocyte deportation during inflammation. Therefore, CD11b and CD18 will be expressed on the surface of neutrophils, and analyzed by flow cytometry, the results show that britinib can significantly reduce the expression of CD11b and CD18 in fMLF-activated human neutrophils (Figure 8 ). In addition, Brittatinib also inhibited IL-8-induced CD11b expression in human neutrophils (data not shown).

Experimental example 2 Brittatinib reduces lipopolysaccharide (lipopolysaccharide, LPS) induced acute lung injury in mice (ALI) and mortality

This example discusses the anti-inflammatory effect of britinib in vivo. To this end, BALB/c mice were administered britinib (25 mg/kg) or DMSO by intraperitoneal injection, followed by intratracheal spraying of LPS for 5 hours. External photographs of the lungs and HE-stained histopathological sections showed that LPS induced tissue hemorrhage and erythema, thickening of alveolar septa, and interstitial edema (Figure 9). Furthermore, inflammatory MPO and Ly6G positive cells (specific markers of neutrophils), protease release (NE), cytokine production (IL-1β), oxidative stress-induced lipid peroxidation were observed after LPS administration (4-HNE) and vascular permeability (clasticin) infiltration. Distortion of the lung structure was significantly suppressed in the britinib-treated group. In addition, britinib also attenuated LPS-activated Vav phosphorylation (p-Vav) (Figure 9).

MPO activity and total protein levels in the lung tissue of mice, representing the severity of pulmonary edema, were significantly increased in the control group, but significantly improved in the britinib-treated group (Fig. 10, (A) and (B) )). In addition, the amount of LPS-induced NET formation (Ly6G ⁺ citH3 ⁺ cell accumulation) was also significantly decreased after Brittatinib treatment (Fig. 10, (C)).

In LPS-treated mice, it was further observed that Brittatinib increased the survival rate of mice. BALB/c mice were injected with LPS (5 mg/Kg) and their survival was monitored for 5 days. All mice in the control group died within 2 days, while the survival time of each britinib-treated mouse (25 mg/Kg) was significantly prolonged (log-rank test, p = 0.0085; Fig. 11).

Although the specific embodiments of the present invention have been disclosed in the above embodiments, they are not intended to limit the present invention. Those who have ordinary knowledge in the technical field of the present invention, without departing from the principle and spirit of the present invention, when Various alterations and modifications can be made to it, so the protection scope of the present invention should be defined by the appended patent scope.

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

Figure 1 shows that Brittatinib inhibits superoxide anion production in activated human neutrophils. Incubate neutrophils (6 × 10 ⁵ cells/mL) with 0.1% DMSO or 0.3–10 μM britinib for 5 minutes, and then expose them to various stimuli that can activate them and produce superoxide anions , including (A) fMLF, (B) MMK-1, (C) NaF, or (D) PMA, followed by the measurement of superoxide anion content using the ferrochrome reduction method. (E) Cytotoxicity was assessed using LDH release. (F) In a cell-free xanthine/xanthine oxidase system, the ability of britinib to scavenge superoxide was evaluated by measuring the reduction degree of WST-1 at 450 nm wavelength with a spectrometer at 20 U/mL Superoxide dismutase was used as a positive control. All data are presented as box plots, median (min-max; n = 6). * p < 0.05 for all data compared with DMSO + fMLF group.

Figure 2 shows that Brittatinib reduces ROS generation in neutrophils activated by fMLF. (A) Human neutrophils labeled with DHR123 were pretreated with 0.1% DMSO or 0.1-3 μM Brittatinib, then activated with 0.1 μM fMLF and monitored by flow cytometry. ROS levels in neutrophils and mean fluorescence intensity (MFI) of DHR123 are presented in box plots with median values (minimum-maximum; n = 5). (B) Neutrophils were incubated with 0.1–3 μM britinib or 0.1% DMSO with or without 0.1 μM fMLF for 5 minutes, followed by real-time monitoring of chemiluminescence using an ELISA reader The change. The peak value of chemiluminescence is presented as a box plot, median value (minimum-maximum; n = 5; right panel). * p < 0.05 for all data compared with DMSO + fMLF group.

Figure 3 shows that Brittatinib attenuates the amount of NE released from activated neutrophils. Neutrophils were treated with 0.1–10 μM britinib or 0.1% DMSO for 5 minutes, and co-induced with (A) fMLF, (B) MMK-1, (C) NaF or (D) LTB4 with NE substrate NE was generated and the amount of released NE was assessed spectroscopically. Data are presented as box plots, median (min-max; n = 6). * p < 0.05 for all data compared with DMSO + fMLF group.

Figure 4 shows that Brittatinib reduces NET production in PMA-activated neutrophils. Human neutrophils were pretreated with 0.1% DMSO or 1-10 μM Brittatinib for 10 min and then incubated with or without 10 nM PMA. (A) Use Sytox green (a nucleic acid stain) to quantify the generated NETs; (B) Stain neutrophils with anti-NE (red) or anti-MPO (green) antibodies, and then use a confocal microscope For analysis, DNA was detected using Hoechst 33342 (blue); (C) Scanning electron micrograph of neutrophils. Shown are representative images. Data are presented as box plots, median (min-max; n = 5). * p < 0.05 for all data compared with DMSO + fMLF group.

Figures 5A to 5E show that Brittatinib inhibits the extent to which ERK and SFK are phosphorylated in fMLF-activated neutrophils. Neutrophils were pre-incubated with 0.1% DMSO or 10 μM britinib, and then stimulated with 0.1 μM fMLF. (A) SFK, (B) Akt (S473), (C) ERK, (D) JNK, and (E) p38 phosphorylated and total immunoblots and their associated quantitative data are presented in box plots, middle Number of bits (min-max; n = 6). * p < 0.05 for all data compared with DMSO + fMLF group.

Figures 6A to 6F show that britinib can inhibit the phosphorylation of SFKs. The degree of phosphorylation of (A) Src, (B) Lyn, (C) Fgr and (D) Hck, and downstream proteins (E) Btk and (F) Vav were determined by immunoblotting method.

Figures 6G to 6J show that Brittatinib inhibits the activity of multiple enzymes. The enzyme activity of (G) Src, (H) Lyn, (I) Fgr or (J) Hck (1.5 ng/mL) was evaluated with ADP-Glo Kinase Assay Kit. After 125 μM ATP (substrate) was added to the reaction mixture after incubation alone with tinib or 0.1–3 μM PP2, enzyme activity was measured 60 min later. Data are presented as box plots, median (min-max; n = 6). * p < 0.05 for all data compared with DMSO + fMLF group.

Figure 7 shows that Brittatinib inhibits the adhesion and migration of fMLF-activated human neutrophils. Hoechst 33342-labeled neutrophils (10 ⁶ cells/mL) were treated with 0.1% DMSO or 1-10 μM Brittatinib for 5 min, followed by 5 min with or without 0.1 μM fMLF/1 μg/mL CB minute. Incubate neutrophils with bEnd.3 cells at 37°C for 30 minutes. The number of neutrophils attached to the bEnd.3 cells was observed and counted under a fluorescent microscope. (A) Amount of attached neutrophils; (B) Human neutrophils were treated with DMSO or 1-10 μM britinib on top of the chemoattractant chamber for 5 min and then treated with or without 0.1 μM fMLF After another 60 minutes of treatment, the number of migrated neutrophils was measured with a cell counter. Data are presented as box plots, median (min-max; n = 5). * p < 0.05 for all data compared with DMSO + fMLF group.

Figure 8 shows that Brittatinib reduces the expression of CD11b (integrin α _M ) and CD18 (integrin β ₂ ) in fMLF-activated neutrophils. Neutrophils were incubated with 0.1% DMSO or 1-10 μM Brittatinib for 5 min and then treated with or without 0.1 μM fMLF/1 μg/mL CB for 5 min. The mean fluorescence intensity (MFI) of FITC-labeled antibody against (A) CD11b and (B) CD18 was detected by flow cytometry. Data are presented as box-and-whisker plots, median (min-max; n = 6). * p < 0.05 for all data compared with DMSO + fMLF group.

Figure 9 shows that Brittatinib attenuates LPS-induced ALI in mice. BALB/c mice (n = 6 per group) were injected with vehicle (10% DMSO) or 25 mg/kg britinib by intraperitoneal injection, followed by LPS by intratracheal spray for 5 hours. (A) Light microscope image of the outside of the lung, and (B) H&E stained, Ly6G-positive, MPO-positive, NE-positive, IL-1β-positive, 4-HNE-positive, cladin-positive, and p-Vav-positive lung sections Light microscope image.

Figure 10 shows that Brittatinib reduces LPS-induced NET production in mice. BALB/c mice (n = 6 per group) were injected with vehicle (10% DMSO) or 25 mg/kg britinib by intraperitoneal injection, followed by LPS by intratracheal spray for 5 hours. (A) MPO activity and (B) total protein concentration in lung tissue measured 5 h after LPS spraying. (C) Immunofluorescence images of DAPI-positive, Ly6G-positive and citH3-positive lung sections. * p < 0.05 when compared with the vehicle+LPS group; # p < 0.05 when compared with the vehicle alone group.

Figure 11 Brittatinib reduces the mortality of LPS-induced mice. BALB/c mice (n = 6 per group) were injected intraperitoneally with vehicle (10% DMSO) or 25 mg/kg britinib followed by intraperitoneal injection of LPS. Mouse survival was monitored for 5 days. Data are presented as box plots, median (min-max; n = 6). All data are * p < 0.05 when compared with the vehicle + LPS group, # p < 0.05 when compared with the vehicle alone group.

Claims (6)

The invention relates to a use of britinib, which is suitable for preparing a medicine for treating acute lung injury. The use as described in claim 1, wherein the content of the britinib in the therapeutic drug is between 0.1 mg and 60 g. The use as described in Claim 1, wherein the acute lung injury is acute respiratory distress syndrome (acute respiratory distress syndrome, ARDS). The use as described in claim 3, wherein the ARDS is blood transfusion-related lung injury, ventilator-induced lung injury, bacteria-induced lung injury or virus-induced lung injury. The use as described in claim 1, wherein the patient is a mammal. The use as described in claim 1, wherein the patient is a human being.

Images