TWI843149B - A formyl peptide receptor 1 antagonist and uses thereof - Google Patents

Abstract

Disclosed herein is related to a novel formyl peptide receptor 1 (FPR1) antagonist having a chemical structure of formula (I), and its uses in manufacturing a medicament for the treatment and/or prophylaxis of diseases mediated by FPR1 ( e.g.,acute respiratory distress syndrome, ARDS).

Description

A type I methyl peptide receptor antagonist and its use

The present disclosure generally relates to the technical field of lung disease treatment, and more particularly to a type I formyl peptide receptor 1 (FPR1) antagonist. The present disclosure also covers the use of the antagonist in the preparation of a drug that can treat and/or prevent diseases mediated by FPR1.

In recent years, acute respiratory distress syndrome (ARDS) has gradually received attention from the medical community in the field of clinical critical care medicine. Although modern medical standards are constantly improving and medical equipment can provide patients with critical respiratory conditions with the most advanced oxygen supply equipment, artificial respirators, and drugs for treatment, the mortality rate of ARDS patients is still high, as high as 40-50%. At the same time, when patients are admitted to the intensive care unit due to respiratory failure and multiple organ failure, it often consumes considerable medical manpower and medical resources. Therefore, ARDS is a very difficult disease in the field of critical care medicine. There is currently no effective treatment for ARDS. Current treatments are still mainly supportive, including the use of artificial respirators, hemodialysis to treat organ failure, or the administration of steroids, but their effectiveness is very limited.

ARDS was first reported in 1967. Patients with ARDS have some common clinical features, including acute hypoxic respiratory failure, severe infiltration of both lungs on chest X-ray, and very high mortality, although the patient's hypoxia responds to positive end-expiratory pressure (PEEP) therapy used by the ventilator. The causes of ARDS vary, such as severe lung infection (pneumonia) or systemic infection (sepsis), trauma, multiple blood transfusions, severe burns, severe pancreatitis, drowning or other aspiration events, drug reactions, or the use of large amounts of fluids during the recovery period after lung trauma, which may induce lung damage and lead to ARDS. In microscopic pathological examination, in the early stage of ARDS, obvious macrophage and neutrophil infiltration can be seen in the lung tissue. In the middle and late stages of ARDS, some cells in the lung (such as type II alveolar cells, fibroblasts, and immune cells) increase significantly, which will lead to fibrosis in the lungs, resulting in loss of lung gas exchange function, so that patients cannot be weaned from the ventilator. Generally speaking, ARDS often occurs within 72 hours after an acute lung injury event. At this time, although the direct damage to the lungs may have disappeared, the activated white blood cells continue to destroy the lung tissue.

On the surface of these activated leukocytes (including macrophages, neutrophils, eosinophils, and basophils), a group of molecules called formyl peptide receptors (FPRs) are found to be overexpressed. The FPRs can recognize N -formyl peptides and belong to a type of G protein-coupled receptor ( GPCR ). Human FPRs can be roughly divided into three types: type I FPR (FPR1), type II FPR (FPR2), and type III FPR (FPR3). Among them, FPR1 is relatively important. It has been reported that FPR1 is an important regulatory factor in the recruitment of neutrophils and the generation of pulmonary fibrosis. Therefore, an appropriate FPR1 antagonist is considered to have the potential to be developed into a drug for the treatment of immune-related diseases (such as the aforementioned ARDS), thereby improving the current treatment of ARDS.

In view of this, the relevant field urgently needs to develop a novel drug that can act through FPR1, so as to develop a safe and effective treatment method for treating and/or preventing immune-related diseases and/or disorders.

The content of the invention is intended to provide a simplified summary of the present disclosure so that readers can have a basic understanding of the present disclosure. This content of the invention is not a complete overview of the present disclosure, and it is not intended to point out the important/key elements of the embodiments of the present invention or to define the scope of the present invention.

The present disclosure is based at least in part on the unexpected discovery by the inventors that a compound (having the structural formula (I)) isolated from a secondary metabolite of a marine Bacillus sp. has a strong regulatory effect on a receptor molecule FPR1. The compound can compete with an FPR1 ligand (also an FPR1 agonist) for binding to FPR1, thereby antagonizing the FPR1 agonist's signaling to FPR1, and can be used to develop a drug that can treat diseases mediated by FPR1, particularly a drug that can treat ARDS mediated by FPR1.

Accordingly, one aspect of the present disclosure relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament, wherein the medicament is administered in an effective amount for treating and/or preventing acute respiratory distress syndrome (ARDS) in a subject in need of treatment: Wherein, R ₁ is a C _6-12 linear or branched alkyl group.

According to the implementation mode of the present disclosure, the compound is a _C10 branched alkyl. According to a specific working example of the present disclosure, the formula (I) has the chemical structure of formula (Ia): (Ia).

According to another specific working embodiment of the present disclosure, the formula (I) has the chemical structure of formula (Ib): (Ib).

As described above, the compound of formula (I) or its pharmaceutically acceptable salt in the present disclosure is an FPR1 antagonist. According to the embodiment of the present disclosure, the compound of formula (I) in the present disclosure is a biologically active secondary metabolite extracted from a marine Bacillus sporogenes.

According to a preferred embodiment of the present disclosure, the effective amount of the compound of formula (I) or its pharmaceutically acceptable salt is about 0.01-100 mg/kg; more preferably, the effective amount of the compound of formula (I) or its pharmaceutically acceptable salt is about 0.1-1 mg/kg.

According to the preferred embodiment of the present invention, the individual in need of treatment is a human.

After reading the following implementation methods, a person with ordinary knowledge in the technical field to which the present invention belongs can easily understand the basic spirit and other invention purposes 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 disclosure more detailed and complete, the following provides an illustrative description of the implementation and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The implementation method covers the features of multiple specific embodiments and the method steps and their sequences for constructing and operating these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and step sequences.

I. Definition

For convenience, specific terms used in this specification, embodiments and the attached patent claims are collected here. Unless otherwise defined in this specification, the scientific and technical terms used herein have the same meaning as those understood and used by ordinary knowledgeable persons in the technical field to which the present invention belongs. In addition, where there is no conflict with the context, singular terms used in this specification include plural forms of the terms, and plural terms used also include singular forms of the terms. Specifically, in this specification and the patent claims, the singular forms "a", "an" and "the" include plural references, unless otherwise indicated by the context. In addition, in this specification and the patent claims, the expressions "at least one" and "one or more" have the same meaning, and both represent one, two, three or more. Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of molecular biology, synthetic chemistry, structural biology and immunology, which are within the skill of the art. Such techniques are described in detail in the public literature.

Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate, the relevant numerical values in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations caused by individual testing methods. Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range. Alternatively, the word "about" means that the actual value falls within the acceptable standard error of the mean, depending on the consideration of a person of ordinary skill in the art to which the present invention belongs. Except for experimental examples, or unless otherwise expressly stated, it is understood that all ranges, quantities, values and percentages used herein (for example, to describe material usage, time duration, temperature, operating conditions, quantity ratios and the like) are modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the attached patent application are approximate values and can be changed as needed. At least, these numerical parameters should be understood as the indicated number of significant digits and the values obtained by applying the general rounding method. Herein, the numerical range is expressed from one end point to another or between two end points; unless otherwise stated, the numerical range described herein includes the end points.

As used herein, the term "an effective amount" refers to an effective amount that, in the necessary dosage and time, can achieve the desired therapeutic result for cancer treatment. For example, in the treatment of cancer, the recombinant polypeptides, complexes, immunocompositions, and methods disclosed herein will effectively prevent cancer cell proliferation and/or growth. An effective amount of an agent does not necessarily cure the disease or condition, but will provide treatment for the disease or condition, thereby delaying, hindering or preventing the onset of the disease or condition, or alleviating the symptoms of the disease or condition. The specific effective amount or sufficient amount will vary depending on a variety of factors, such as the specific condition to be treated, the physical condition of the patient (e.g., the patient's weight, age or sex), the type of mammal or animal to be treated, the duration of treatment, the nature of concurrent treatment (if any), and the specific formulation used. An effective amount can be expressed, for example, as the total mass of active agent (e.g., in grams, milligrams or micrograms), or as the ratio of the mass of active agent to body weight (e.g., in milligrams per kilogram (mg/kg)). An effective amount can be divided into one, two or more doses in an appropriate form for administration once, twice or more times throughout a specified period. Preferably, an effective amount refers to a human equivalent dose (HED), which is the maximum safe dose for human subjects. HED can be estimated according to the industry guidance "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers" issued by the U.S. Food and Drug Administration (FDA).

Unless otherwise indicated, a "therapeutically effective amount" of a compound means an amount sufficient to provide a therapeutic benefit for the treatment or management of a disease, or to delay or minimize one or more symptoms associated with the disease. A therapeutically effective amount of a compound refers to an amount of a therapeutic agent used alone or in combination with other drugs that can be used to treat or manage a disease to provide a therapeutic benefit. The term "therapeutically effective amount" may also refer to an amount sufficient to enhance the overall therapeutic effect or reduce, prevent symptoms, or enhance the efficacy of other therapeutic agents.

Unless otherwise indicated, a "prophylactically effective amount" of a compound is an amount sufficient to prevent the occurrence or recurrence of a disease or condition, or one or more symptoms associated with the disease or condition. A "prophylactically effective amount" of a compound is an amount of a therapeutic agent used alone or in combination with other drugs that can be used to prevent the occurrence of a disease and provide a prophylactic benefit. The term "prophylactically effective amount" may also refer to an amount sufficient to enhance the overall prophylactic effect or enhance the efficacy of other prophylactic agents.

As used herein, the term "treat", "treatment" or "treating" may refer to a curative or palliative measure. Specifically, the term "treating" as used herein refers to administering a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition or method containing the compound of formula (I) to an individual, wherein the individual suffers from ARDS, has symptoms associated with ARDS, or has a disease or condition secondary to ARDS, etc., in order to partially or completely reduce, improve, alleviate, delay the onset, inhibit the progression of the disease, reduce the severity, and/or reduce the incidence of one or more symptoms or signs of ARDS.

The term "subject" or "patient" refers to an animal, including human species, that can be treated with the compounds of formula (I) or pharmaceutically acceptable salts thereof, or pharmaceutical compositions or methods containing the compounds of formula (I) disclosed herein. Unless a gender is explicitly indicated, the term "subject" or "patient" means both male and female. Accordingly, the term "subject" or "patient" includes any mammal that may benefit from ARDS treatment. Exemplary "subjects" or "patients" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds, and poultry. In an exemplary embodiment, the subject is a mouse. In another exemplary embodiment, the subject is a human.

The terms "administered", "administering" or "administration" are used interchangeably in the present disclosure to refer to the administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition or method containing the compound of formula (I) of the present disclosure to a subject in need thereof.

II. Invention details

Accordingly, the present disclosure is about a compound extracted from a secondary metabolite of a marine bacillus, having a chemical structure of formula (I), which has a strong regulatory effect on FPR1. It competes with FPR1 agonists for binding to FPR1, thereby blocking the activation of FPR1 by FPR1 agonists, thereby achieving the purpose of inhibiting FPR1 activation, and can be used to treat diseases related to FPR1 activation, especially ARDS related to FPR1 activation. Therefore, the compound of the present disclosure is a potential FPR1 antagonist, which can be used to develop a lead compound that can treat or prevent diseases and/or disorders mediated by FPR1 (e.g., ARDS).

1. Treatment

Accordingly, one aspect of the present disclosure is a method for treating and/or preventing a subject suffering from or suspected of suffering from a disease and/or disorder mediated by FPR1. The method of the present disclosure comprises administering to the subject a therapeutically effective amount or a prophylactically effective amount of a compound of formula (I) to reduce, alleviate, and/or prevent the symptoms of the disease and/or disorder mediated by FPR1: .

The present disclosure also encompasses a method for treating or preventing diseases and/or disorders mediated by FPR1, comprising administering to a subject a therapeutically effective amount or a prophylactically effective amount of a compound of formula (I) to reduce, alleviate, and/or prevent symptoms of the disease and/or disorder mediated by FPR1.

Specifically, in the above-mentioned compound of formula (I), R ₁ is a straight or branched saturated alkyl group (alkyl) having 6 to 12 carbon atoms ("C _6-12 alkyl" (C _6-12 alkyl)). In certain embodiments, R ₁ is a straight or branched alkyl group having 6 carbon atoms ("C ₆ alkyl" (C ₆ alkyl)). In certain embodiments, R ₁ is a straight or branched alkyl group having 7 carbon atoms ("C ₇ alkyl" (C ₇ alkyl)). In certain embodiments, R ₁ is a straight or branched alkyl group having 8 carbon atoms ("C ₈ alkyl" (C ₈ alkyl)). In certain embodiments, R ₁ is a straight or branched alkyl group having 9 carbon atoms ("C ₉ alkyl" (C ₉ alkyl)). In certain embodiments, R ₁ is a straight or branched alkyl group having 10 carbon atoms ("C ₁₀ alkyl"). In certain embodiments, R ₁ is a straight or branched alkyl group having 11 carbon atoms ("C ₁₁ alkyl"). In certain embodiments, R ₁ is a straight or branched _alkyl group having ₁₂ carbon atoms (" _{C 12 alkyl} ").

Exemplary C _6-12 alkyl groups include, but are not limited to, hexane (C ₆ , hexane) (e.g., n-hexane, 2-methylpentane, 3-methylpentane, 2,3-dimethylbutane, 2,2-dimethylbutane); heptane (C ₇ , heptane) (e.g., n-heptane, 2-methylhexane, 3-methylhexane, 2,2-dimethylpentane, 2,3-dimethylpentane, 2,4-dimethylpentane, 3,3-dimethylpentane, 3-ethylpentane, 2,2,3-trimethylbutane); octane (C ₈ octane) (e.g., 2-methylheptane, 3-methylheptane, 4-methylheptane, 2,2-dimethylhexane (neo-octane), 3,3-dimethylhexane, 3-ethylhexane, 2,3-dimethylhexane, 2,4-dimethylhexane, 2,5-dimethylhexane, 3,4-dimethylhexane, 3,4-dimethylhexane, 3,4-dimethylhexane, 3,4-dimethylhexane, 3,4-dimethylhexane, 3,4-dimethylhexane, 3,4-dimethylhexane, 3,4-dimethylhexane, 3,4-dimethylhexane, 3,3 ... thylhexane), 2,3,4-trimethylpentane, 2,2,3-trimethylpentane, 2,2,3-trimethylpentane, 2,2,4-trimethylpentane; isooctane), 2,3,3-trimethylpentane, 2-methyl-3-ethylpentane, 3-methyl-3-ethylpentane, 2,2,3,3-tetramethylbutane); nonane (C ₉ , nonane) (e.g., n-nonane); decane (C ₁₀ , decane) (e.g., n-decane, 2-methylnonane, 3-methylnonane); undecane (C ₁₁ , hendecane) (e.g., n-hendecane); or dodecane (C ₁₂ , dodecane) (e.g., n-dodecane).

According to one embodiment of the present disclosure, R ₁ is 3-methylnonane (IA-1), and the compound of formula (I) of the present disclosure has the following chemical structure (Ia): (Ia).

According to another specific embodiment of the present disclosure, R ₁ is 2-methylnonane (IA-2), and the compound of formula (I) of the present disclosure has the chemical structure of the following formula (Ib): (Ib).

Unless otherwise indicated, the alkyl group in each embodiment may be unsubstituted, i.e., "unsubstituted alkyl", or substituted with one or more substituents (e.g., halogens such as fluorine or -OH), i.e., "substituted alkyl". In certain embodiments, _R1 is an unsubstituted _C6-12 alkyl group (e.g., an unsubstituted C10 alkyl group). In certain embodiments, _R1 is a substituted _C6-12 alkyl group (e.g., _{a C10} alkyl group substituted with, for example, _-CF3 or _-CH2OH ).

The compound of formula (I) disclosed herein can be extracted from a secondary metabolite (culture medium) of a marine Bacillus by the method disclosed in the working examples of the disclosure. In addition, the compound of formula (I) disclosed herein contains one or more stereocenters, and thus the compound of formula (I) disclosed herein can exist in the form of a racemic mixture of mirror-image compounds or a mixture of non-mirror-image compounds. Therefore, the disclosure also includes such compounds in the form of pure stereoisomers, and mixtures thereof. Stereoisomers can be synthesized in an asymmetric manner, or purified by separating them from each other using crystallization, chromatography or a resolving agent. A preferred method for separating mirror-image compounds is preparative HPLC. Alternatively, the racemic mixture can be reacted with an optically active separating agent in the presence of a suitable solvent to separate the mirror image compounds from each other. Depending on the optical activity of the separating agent, one of the two mirror image compounds can be separated as a highly optically active insoluble salt, while the other mirror image compound remains in solution.

Therefore, the present disclosure further encompasses a stereochemical mixture of the compound of formula (I); it also includes the structural isomers (i.e., cis- or trans-isomers) of the compound of formula (I) of the present disclosure, and whether the compound exists in the form of a pure compound or in the form of a mixture of stereoisomers, it is also within the scope of the present disclosure.

According to the embodiments of the present disclosure, the compound of formula (I) (including compounds of formula (I-a) or (I-b)) is an FPR1 antagonist, and the disease and/or disorder mediated by FPR1 may be ARDS.

The methods of the present disclosure also encompass the use of other known drugs or therapies for treating diseases and/or disorders mediated by FPR1 (e.g., ARDS), i.e., administering another known drug or therapy useful for treating ARDS before, simultaneously with, or after administering a compound of formula (I) of the present disclosure to a subject in need of treatment. The known drug may be a known FPR1 antagonist (e.g., cyclosporine A, cyclosporine H, N-(N-aroyl-L-tryptophanyl)-D-phenylalanine, 3,4-dihydroisoquinolin-2(1H)-yl-3-phenylurea, etc.); a known ARDS treatment drug (e.g., corticosteroids (e.g., hydrocortisone, triamcinolone acetonide, dexamethasone, halometasone, etc.), a surfactant, N -acetylcysteine ( N -acetylcysteine), statins (e.g., fluvastatin, atorvastatin, cerivastatin, pitavastatin, simvastatin, etc.), β-agonists (e.g., dobutamine, clenbuterol, salbutamol, methoxyphenamine, etc.); known anti-inflammatory drugs (e.g., alcofenac, aceclofenac, sulindac, tolmetin, etodolac, fenopren, thiaprofenic acid, meclofenamic acid, etc.); acid, meloxicam, tenoxicam, lornoxicam, nabumetone, acetaminophen, phenacetin, ethenzamide, sulpyrine, mefanamic acid, flufenamic acid, diclofenac sodium, loxoprofen sodium, phenylbutazone, indomethacin, ibuprofen, ketoprofen, etc.); the known treatment is a respiratory therapy, for example, using a respirator, high frequency ventilation, extra-corporeal membrane oxygenation, liquid or partial liquid ventilation. For example, in the process of treating ARDS, in addition to administering the compound of formula (I) of the present disclosure to the individual in need of treatment, the method of the present disclosure further comprises administering a respiratory therapy and/or other known ARDS therapeutic drugs to the individual.

According to the preferred embodiment of the present invention, the compound of formula (I) is administered to the subject in need of treatment at an amount of about 0.01-100 mg/kg, for example, about 0.01, 0.05, 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 1, 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, or 100 mg/kg; preferably about 0.1-50 mg/kg The dosage is administered to the subject in need of treatment, for example, about 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, or 50 mg/kg; more preferably, about 0.1-20 mg/kg is administered to the subject in need of treatment, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 10.10, 11.11, 12.13, 14.15, 16.17, 18.19, 20.21. 9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17 .5, 18, 18.5, 19, 19.5, or 20 mg/kg; more preferably, about 0.1-10 mg/kg is administered to the subject in need of treatment, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2. Preferably, the compound of formula (I) is administered to the subject in need of treatment in an amount of about 0.5 mg/kg. Preferably, the compound of formula (I) is administered to the subject in need of treatment in an amount of about 1 mg/kg. Preferably, the compound of formula (I) is administered to the subject in need of treatment in an amount of about 0.1-1 mg/kg, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/kg. According to one embodiment of the present disclosure, the compound of formula (I) is administered to the subject in need of treatment in an amount of about 0.5 mg/kg. According to another embodiment of the present disclosure, the compound of formula (I) is administered to the subject in need of treatment in an amount of about 1 mg/kg.

The compounds of formula (I) disclosed herein can be administered by appropriate routes known to those skilled in the art, such as oral, transmucosal (e.g., nasal, sublingual, intrabuccal, vaginal, rectal) or transdermal, or injection (e.g., intradermal, subcutaneous, intravenous, intramuscular, intraarterial) routes. In general, the most appropriate route of administration will vary depending on various factors, such as the nature of the drug (e.g., its stability in the blood circulation), and/or the physiological conditions of the individual (e.g., whether the individual has tolerance to intravenous administration).

It is understood that medical personnel can determine the exact dosage of the compound of formula (I) disclosed herein within the scope of reasonable medical judgment to achieve a certain medical effect. The effective amount administered to a particular individual will depend on various factors, including the disease to be treated and the severity of the disease; the activity of the specific active ingredient used; the composition used; the species, age, weight, general health condition, sex and dietary habits of the individual, the severity of side effects or drug abnormalities; the time of administration, route of administration and the rate of excretion of the active ingredient; the duration of treatment; and other factors known in the medical field. At the same time, an effective amount can be covered in a single dose (e.g., a single intravenous injection) or multiple doses (e.g., multiple intravenous injections). When multiple doses are administered to an individual, the frequency of administering the multiple doses to the individual can also be adjusted according to actual circumstances. The above factors and adjustment methods are well known and commonly used by those with ordinary knowledge in this field.

The subject to be treated using the methods of the present disclosure is a mammal, preferably, the subject is a human.

2. Pharmaceutical compositions

The present disclosure also encompasses providing a pharmaceutical composition for treating diseases and/or disorders mediated by FPR1. The pharmaceutical composition comprises an effective amount of a compound of formula (I) and a pharmaceutically acceptable carrier.

Generally speaking, if the total weight of the pharmaceutical composition is taken as 100%, the compound of formula (I) accounts for about 0.01-99% (weight %) of the total weight of the pharmaceutical composition; in certain embodiments, the compound of formula (I) accounts for at least 0.1% (weight %) of the total weight of the pharmaceutical composition, and in specific embodiments, the compound of formula (I) accounts for at least 5% (weight %) of the total weight of the pharmaceutical composition; in other embodiments, the compound of formula (I) accounts for at least 10% (weight %) of the total weight of the pharmaceutical composition; in another embodiment, the compound of formula (I) accounts for at least 25% (weight %) of the total weight of the pharmaceutical composition.

In certain embodiments, the pharmaceutical composition disclosed herein further comprises a known FPR1 antagonist. According to another embodiment of the present invention, the pharmaceutical composition further comprises a known ARDS therapeutic drug. According to yet another embodiment of the present invention, the pharmaceutical composition further comprises an anti-inflammatory drug.

Pharmaceutically acceptable carriers are those ingredients that are compatible with other ingredients in the pharmaceutical composition formulation and are biologically acceptable. The pharmaceutical compositions of the present disclosure containing the compound of formula (I) can be formulated into dosage forms suitable for oral, transmucosal or transdermal administration, or injection. Examples of dosage forms include, but are not limited to, tablets (chewable), half-tablets, capsules (e.g., gelatin capsules), cachets, lozenges, suspensions, suppositories, ointments, salves, pastes, powders, dressings, plasters, solutions, patches, sprays (e.g., nasal sprays or inhalants), gels; liquid dosage forms suitable for oral or transmucosal administration (e.g., suspensions (aqueous or oily suspensions), emulsions (oil-in-water or water-in-oil emulsions), solutions or elixirs); liquid dosage forms suitable for injection; or sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted into liquid dosage forms suitable for injection.

The formulation of the pharmaceutical composition disclosed herein is suitable for administration by a variety of different routes. For example, a coating (or enteric coating) that delays disintegration and absorption in the gastrointestinal tract can be coated on the outside of the tablet using known techniques to achieve a sustained release effect over a longer period of time (i.e., a long-acting dosage form). For example, a substance that delays release, such as monostearate or distearate, can be added. Alternatively, a suitable coating can be coated on the outside of the tablet to form a permeable therapeutic agent. Similarly, the compounds of the present invention can also be made into liposomes to prevent them from being destroyed by degradative enzymes, to help them be transported in the circulatory system and to effectively pass the drug through the cell membrane and deliver it into the cell.

Solubilizers, emulsifiers, surfactants (e.g., cyclodextrin (α-cyclodextrin or β-cyclodextrin)), non-aqueous solvents (including, but not limited to, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, ethylene glycol, 1,3-butyl glycol, dimethylformamide, dimethyl sulfoxide (DMSO), biocompatible oils (e.g., cottonseed oil, nut oil, corn oil, olive oil, rapeseed oil, sesame oil), glycerol, tetrahydrofuranol, polyethylene glycol, fatty acid esters of sorbitol, and mixtures thereof (e.g., a mixture of DMSO and sesame oil)) can also be used to encapsulate poorly water-soluble drugs into liquid dosage forms.

The composition, shape and form of a dosage form may vary depending on the route of administration. For example, a dosage form for treating an acute condition may contain a higher amount of active agent than a dosage form for treating a chronic condition; similarly, an injectable dosage form may contain a smaller amount of active agent than an oral dosage form. Such adjustments in dosage or carrier for different dosage forms are within the knowledge of those skilled in the art.

2.1 Oral dosage form

The oral dosage form of the pharmaceutical composition disclosed herein can be a tablet, a half-tablet, a capsule, or a liquid (e.g., syrup) dosage form. Such dosage forms contain a predetermined amount of active ingredient and can be manufactured by methods well known in the art, which have been described in detail in existing public documents. Typical oral dosage forms are manufactured by mixing the active ingredient with at least one excipient in accordance with traditional pharmaceutical preparation methods, wherein the choice of excipient can be determined according to the preparation form. Since oral administration is the easiest way to use, tablets and capsules are the most common forms. If necessary, a standard solution or non-solution coating method can be used to coat the outside with an appropriate coating. Generally speaking, the active ingredient can be uniformly mixed with a liquid carrier, a solid carrier, or both, and then molded into an appropriate shape for manufacturing. A disintegrant may also be added to help it dissolve quickly. Or a lubricant may be added to help its manufacturing.

2.2 Transmucosal or transdermal dosage forms

Transmucosal or transdermal dosage forms include, but are not limited to, ophthalmic solutions, sprays, creams, lotions, ointments, gels, solutions, emulsions, and other known dosage forms. Formulations that can be used for transmucosal or transdermal dosage forms are well known in the art. In addition, depending on the specific tissue to be treated, other transdermal enhancers (i.e., agents used to help deliver active agents through the skin or mucous membranes) can also be used simultaneously or successively. In addition, the pH value of the pharmaceutical composition, the polarity, ionic strength or tension of the solvent carrier can also be adjusted depending on the tissue to be applied to improve its delivery effect. For example, stearates can be added to change the lipophilicity or hydrophobicity of the active ingredient to help drug delivery.

2.3 Injection dosage form

Injectable dosage forms are generally aseptically processed, including, but not limited to, ready-to-use injection solutions, solids or powders that can be immediately dissolved in a pharmaceutically acceptable injection carrier (e.g., saline), or emulsions. Pharmaceutically acceptable injection carriers are well known in the art, including water, isotonic sodium chloride solution, saline, Ringer's solution, glucose solution; water-miscible carriers, such as ethanol, polyethylene glycol, propylene glycol; non-aqueous solvents, such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, benzyl benzoate.

3. Set

The present disclosure also covers a manufacture or kit comprising a compound of formula (I) of the present disclosure for use in treating or preventing diseases and/or disorders mediated by FPR1 (e.g., ARDS). According to one embodiment, the kit comprises a container for carrying the compound of formula (I) of the present disclosure. Suitable containers may be bottles, vials, syringes, blister packs, etc., and the container material may be glass or plastic. Alternatively, the kit may further comprise a second container for carrying a pharmaceutically acceptable buffer (e.g., saline, Ringer's solution, or glucose solution). Other materials necessary from a commercial or usage perspective may also be included, such as other types of buffers, diluents, fillers, needles or syringes, etc. The kit may further comprise a label affixed to the container or instructions placed in the container.

The kit may also include instructions and guidance for instructing the user on how to use the compound of formula (I) of the present disclosure, or, if necessary, for instructing a second pharmaceutical formulation for treating or preventing diseases and/or disorders mediated by FPR1. In particular, if the kit includes a first formulation (which includes the compound of formula (I) of the present disclosure) and a second formulation, it may further include instructions for the user on how to use the first and second formulations simultaneously, sequentially, or separately. According to one embodiment, the kit includes at least: (1) a first container containing the compound of formula (I) of the present disclosure; and optionally (2) a second container containing a second therapeutic agent, which may be a known FPR1 antagonist, an ARDS treatment drug, or an anti-inflammatory drug; and (3) an instruction manual for use instructing the user on how to use the kit. The instruction manual may be a leaflet, a videotape, a CD, a VCD, or a DVD.

The following are several experimental examples to illustrate certain aspects of the present invention, so as to facilitate the implementation of the present invention by those with ordinary knowledge in the technical field to which the present invention belongs, and these experimental examples should not be regarded as limiting the scope of the present invention. It is believed that after reading the description provided herein, the skilled person can fully utilize and practice the present invention without over-interpretation. All public documents cited herein are regarded as part of this specification in their entirety.

Embodiment

Materials and methods

1. Cell culture

Human neutrophils were isolated from human peripheral blood samples by dextran sedimentation, Ficoll Paque Plus density gradient centrifugation, and erythrocyte hypoosmotic pressure lysis. The isolated human neutrophils were then resuspended in 1 mM calcium Hank’s balanced salt solution (HBSS) containing 2% human serum albumin (HSA), and the cells were counted and adjusted to the required concentration for subsequent experiments.

2. FPR1 Binding test

Human neutrophils contain the receptor for fMLF, i.e., FPR1, on their cell membranes. The analytes (IALB-E, IA-1, or IA-2) compete for receptor binding through the fluorescently labeled fNLFNYK that binds to FPR1. Therefore, receptor binding assays are used to further clarify whether the analytes interact with the receptors. Human neutrophils (2×10 ⁶ cells/mL) were exposed to specific concentrations of analytes (0-5 μM) or fMLF (10 μM) for about 5 minutes at 4°C, and then labeled with or without the FPR1-specific ligand fNLFNYK (2 nM) for 30 minutes. Binding was then immediately measured using a FACScan flow cytometer.

3. Animal testing

LPS was used to induce acute respiratory distress syndrome (ARDS) in mice. BALB/c male mice, approximately 7-8 weeks old, were housed in an animal room with a 12-h light-dark cycle and were given free access to standard chow and water. All animal experimental procedures were in accordance with the guidelines established by the Chang Gung University Animal Care and Use Committee.

The test substance (IALB-E or IA-1) was dissolved in 50 μL of saline containing 10% DMSO and 10% Tween-20, and the final concentration of the test substance was adjusted to 5 mg/kg or 10 mg/kg, and injected into the mice by intravenous injection. One hour later, the mice were anesthetized with Zoletil ^® 50 (containing zolazepam and tiletamine; 30 mg/kg) and xylazine (5 mg/kg), and 2 mg/kg of LPS (Escherichia coli O111:B4; Sigma-Aldrich) was administered intratracheally by spray; the control group mice were administered with saline. Regarding the post-treatment of the test substance IALB-E, 5 mg/kg or 10 mg/kg of IALB-E was injected into the mice through intravenous injection 1 hour after LPS was administered to the mice. Afterwards, 6 hours after LPS was administered to the mice, the mice were sacrificed by cervical dislocation and their lung tissues were removed, fixed with 10% formalin and embedded in paraffin blocks for subsequent tissue section observation.

4. Histopathology and immunohistochemistry analysis

The lung tissue was fixed with 10% formalin and embedded in a paraffin block. After cutting out tissue sections with a thickness of about 5 microns, hematoxylin-eosin (H&E) staining was performed. In terms of immunohistochemical staining, the primary antibody against Ly6g or elastomeric enzyme was first used for identification, followed by secondary antibodies that could recognize the primary antibody. Finally, the tissue was stained with 3,3'-diaminobenzidine (DAB) chromatin and then contrast stained with hematoxylin. In the statistical analysis, the ratio of nuclear staining area to total staining area was calculated for H&E staining area analysis, and the ratio of immunohistochemical positive staining area to total staining area was calculated for Ly6g or elastase staining area analysis.

5. Measuring elastin release

The amount of flexenzyme released was measured using N- methoxysuccinyl-Ala-Ala-Pro-Val- p -nitroanilide (MeOSuc-AAPV-PNA) as the matrix. Briefly, MeOSuc-AAPV-PNA (100 μM) was added to the culture medium, and human neutrophils (6×10 ⁵ cells/mL) were allowed to equilibrate for about 5 minutes before being exposed to a specific concentration of the analyte (IALB-A, IALB-B, IALB-C, IALB-D, IALB-E, IA-1, or IA-2) for about 2 minutes. Next, the cells were treated with cytochalasin B (CB; 0.5 μg/mL) for about 3 minutes, and then activated with specific agonists, including fMLF (0.1 μM), fMMYALF (0.1 μM), MMK1 (0.3 μM), IL-8 (100 ng/mL), LTB ₄ (0.1 μM), or NaF (20 mM), for about 10 minutes or 30 minutes, and the changes in absorbance at a wavelength of 405 nm were measured; the experimental conditions of the IALB-F group were the same, but no agonists were added. In another set of experiments, cells were treated with a specific concentration (1 or 2.5 μM) of the test substance (IA-1 or IA-2), and then activated with a specific concentration (10 ⁰ -10 ⁴ nM) of fMLF. The rest of the steps were the same as above. The experimental results were expressed as the percentage of elastin release.

6. Measuring superoxide anions (O ₂ ^- ) Generation

The amount of O ₂ ^- was determined by the reduction of ferricytochrome c , which is inhibited by superoxide dismutase (SOD). Briefly, human neutrophils (6×10 5 cells/ml or 3×10 5 cells/ml) were treated with the specified concentration of the test substance (IALB-A, IALB-B, IALB-C, IALB-D, IALB-E, IA-1 or IA-2) at 37°C for about 2 minutes after ^0.6 mg/ml of ^{ferricytochrome} c was added to the cell culture medium. Then, the neutrophils were treated with cytochalasin B (1 μg/mL) for about 3 minutes, and then activated with specific agonists, including fMLF (0.1 μM), fMMYALF (0.1 μM), MMK1 (0.3 μM), PMA (0.01 μM), NaF (20 mM), etc., for about 10-30 minutes; the experimental conditions of the IALB-F group were the same, but no agonists were added. The changes in absorbance at 550 nm due to the reduction of ferrocytochrome c were continuously monitored in a dual-beam, 6-slot positioning spectrometer (Hitachi U-3010) with continuous stirring, and the extinction coefficient (ε = 21.1 mM ^-1 /10 mm) was used to calculate the amount of O ₂ ^- generated. In another set of experiments, cells were treated with a specific concentration (1 or 2.5 μM) of the test substance (IA-1 or IA-2), and then activated with a specific concentration (10 ⁰ -10 ⁴ nM) of fMLF. The rest of the steps were the same as above.

7. Measurement CD11b Performance

To measure the expression of CD11b on the cell surface, human neutrophils were mixed in a suspension at 37°C, preheated for 5 minutes, and then treated with a specific concentration (5 μM) of the test substance (IA-1 or IA-2) and then cytochalasin B (1 μg/mL) was added for 3 minutes. Then, specific agonists, including fMLF (0.1 μM), fMMYALF (0.1 μM), and MMK1 (0.3 μM), were added to the cells for 5 minutes and then placed on ice to terminate the reaction. After centrifugation, the supernatant was removed and the cells were resuspended in a balanced salt solution containing 5% bovine serum albumin (BSA). Anti-CD11b antibody labeled with fluorescein isothiocyanate (FITC) was added to react on ice in the dark, and then the balanced salt solution was added to terminate the reaction. Flow cytometry was used to detect the changes in the amount of fluorescence expression to further understand whether the test substance would affect the expression of CD11b on the cell membrane of human neutrophils after stimulation.

8. Measuring intracellular calcium (Ca ²⁺ ) Release amount

Human neutrophils stained with fluo-3/AM (2 µM) were added to the test compound and reacted for 5 minutes, then stimulated with fMMYALF (0.1 µM). Finally, Triton X-100 was added to obtain the maximum fluorescence value (F _max ), and then ethylene glycol tetraacetic acid was added to obtain the minimum fluorescence value (F _min ). The change in fluorescence intensity was recorded by a fluorescence spectrophotometer, and the excitation wavelength and emission wavelength were set to 488 nm and 520 nm, respectively. The change in intracellular Ca ²⁺ concentration was calculated using the following equation: [Ca ²⁺ ] _i (nM) = 400 × (F - F _min ) / (F _max - F), where 400 is the calcium ion dissociation constant of fluo-3 and F is the fluorescence value of the stimulated intracellular calcium ions.

9. Statistical analysis

All experiments were performed at least three times (except for Figures 2A-2D, where the experiments were performed twice in animals treated with IALB-E in LPS-induced ARDS mice), and the experimental results are expressed as mean ± SEM. Statistical analysis was performed using Student's t test, and P < 0.05 was considered statistically significant. All statistical analyses were performed using Sigma Plot software.

Embodiment 1 Extracts from marine Bacillus sporogenes culture fluid have anti-inflammatory effects

1.1 Preparation of extracts from marine Bacillus sporogenes cultures

This example aims to study the therapeutic efficacy of the extract of marine Bacillus culture fluid. For this purpose, the extract of marine Bacillus culture fluid was prepared. After the culture fluid of marine Bacillus was centrifuged to remove bacteria, the supernatant was extracted with ethyl acetate (EA) at room temperature to obtain a crude extract IALB (1.322 g). Then, IALB was filtered with a filter membrane with a pore size of 0.45 μm and then purified by preparative HPLC. The filtrate was loaded onto a silica gel column (Sunniest C18) and eluted with methanol/0.1% formic acid (FA) at a flow rate of 20 ml/min into 6 fractions, namely, the elution fraction of the first fraction (labeled as IALB-A; 28.7 mg), the elution fraction of the second fraction (labeled as IALB-B; 80.1 mg), the elution fraction of the third fraction (labeled as IALB-C; 46.1 mg), the elution fraction of the fourth fraction (labeled as IALB-D; 25.1 mg), the elution fraction of the fifth fraction (labeled as IALB-E; 43.3 mg), and the elution fraction of the sixth fraction (labeled as IALB-F; 359.9 mg). After obtaining the elution fractions of the extracts, the biological activities of the extract fractions were analyzed.

1.2 Characteristic analysis of extracts from the culture medium of marine Bacillus sporogenes

First, we investigated whether the extract fractions had anti-inflammatory effects. Specifically, we investigated the ability of the extract fractions to inhibit superoxide anion production and elastase release by human neutrophils. The cells in the IALB-A-IALB-E group were primed with CB and activated with fMLF after being treated with IALB-A, IALB-B, IALB-C, IALB-D, or IALB-E; and the cells in the IALB-F group were primed with CB only after being treated with IALB-F, but not activated with fMLF. The relevant experimental results are shown in Table 1.

Table 1 Effects of extract fractionation on superoxide anion generation and elastin release Sample (5 μg/mL) Superoxide anion generation Flexase release _IC50a ^​ inhibition% Increase% Significance _IC50a ^​ inhibition% Increase% Significance IALB-A > 10 7.82 ± 2.39 N/ ^A * > 10 1.44 ± 1.57 N/ ^A IALB-B > 10 7.36 ± 1.45 N/ ^A ** > 10 2.03 ± 1.08 N/ ^A IALB-C > 10 13.81 ± 2.52 N/ ^A ** > 10 1.55 ± 2.82 N/ ^A IALB-D > 10 31.81 ± 6.06 N/ ^A * > 10 5.53 ± 4.59 N/ ^A IALB-E 1.60 ± 0.49 97.83 ± 0.82 N/ ^A *** 2.29 ± 0.85 106.58 ± 5.66 N/ ^A *** IALB-F N/ ^A N/ ^A 106.85 ± 7.04 ^c *** N/ ^A N/ ^A 63.25 ± 5.34 ^c *** Note 1: * P <0.05; ** P <0.01; *** P < 0.001, compared with DMSO control group. Note 2: ^a The concentration required to achieve half inhibition concentration (IC ₅₀ ) (μg/mL). Note 3: ^b N/A: indicates not applicable here; ^c The value is promotion: it means that it will not inhibit, but will increase the generation of superoxide anions and the release of elastic enzymes under the action of CB; the value is converted based on the effect of fMLF/CB as 100%.

As shown in Table 1, IALB-E has the ability to strongly inhibit the generation of superoxide anions and the release of elastase by human neutrophils. Therefore, IALB-E was further investigated to confirm whether the mechanism of action of the above ability is related to the signal transmission of FPR1. The experimental results are shown in Figure 1. IALB-E (0-5 μg/ml) showed a significant inhibition of the binding of another FPR1-specific ligand (fNLFNYK) to FPR1 in a dose-dependent manner, indicating that IALB-E has an active ingredient, and the active ingredient is likely to block the activation of human neutrophils through FPR1.

1.3 IALB-E For treatment ARDS Benefits

Based on the above results, IALB-E was further explored using an in vivo animal model to confirm whether IALB-E has therapeutic efficacy for ARDS. The relevant experimental results are shown in Figures 2A-2D. From the immunohistochemical analysis results in Figure 2A, it can be seen that whether IALB-E is administered to mice before LPS stimulation (pretreatment) or after LPS stimulation (posttreatment), both situations can significantly reduce the number of human neutrophils infiltrating lung tissue (whether in the number of nuclear stained cells in H&E staining (Figure 2B) or those with anti-Ly6g reactions (Figure 2C)). At the same time, because the number of human neutrophils is reduced, the release of elastase can be greatly reduced (Figure 2D), thereby protecting the lung tissue from damage. The corresponding staining area statistical analysis results are shown in Figures 2B-2D. The above experimental results clearly demonstrate that IALB-E has the ability to effectively prevent and treat ARDS in mice.

Embodiment 2 IA-1 and IA-2 Has anti-inflammatory properties

2.1 Separate and Identification IA-1 and IA-2

As shown in Example 1, the active ingredient in IALB-E has the ability to strongly inhibit the production of superoxide anions and the release of elastase by human neutrophils. In order to clarify the active ingredient, IALB-E was analyzed by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), thereby obtaining the active ingredient as the following compound of formula (Ia) (i.e., IA-1) and compound of formula (Ib) (i.e., IA-2). At the same time, their structures were confirmed by total ion chromatography (TIC) (data not shown) and mass spectrum (data not shown). (Ia) (Ib)

2.2 IA-1 and IA-2 Effects on the release of elastin

The purpose of this study was to determine the ability of IA-1 and IA-2 to inhibit the release of elastase from human neutrophils. The results are shown in Figures 3A-3F (for IA-1) and 4A-4F (for IA-2).

As shown in Figures 3A-3B, IA-1 can inhibit the release of elastase from human neutrophils activated by exogenous FPR1 agonist fMLF or endogenous FPR1 agonist fMMYALF, with _IC50 of 0.72 ± 0.11 μM and 0.84 ± 0.30 μM, respectively. However, IA-1 has no ability to inhibit the release of elastase from human neutrophils activated by FPR2 agonist MMK1 (Figure 3C), indicating that IA-1 is selective in inhibiting the release of elastase from human neutrophils, and can specifically inhibit the release of elastase caused by activation of cells via the FPR1 (but not FPR2) signaling pathway. In addition, this experiment also explored three other factors that can activate human neutrophils, including IL-8, LTB ₄ , and NaF. These factors will also promote the release of elastic enzymes after activating human neutrophils. The experimental results are shown in Figures 3D-3F. IA-1 also has different degrees of ability to inhibit the release of elastic enzymes, among which the IC ₅₀ of IL-8 is 3.36 ± 0.46 μM. In summary, IA-1 does have the effect of inhibiting the release of elastic enzymes by activated human neutrophils, and therefore has anti-inflammatory effects.

Similar experimental results can also be seen in the experimental results of IA-2 inhibiting the release of elastase from activated human neutrophils. As shown in Figures 4A-4B, IA-2 can inhibit the release of elastase from human neutrophils activated by fMLF or fMMYALF, with IC ₅₀ of 0.85 ± 0.28 μM and 0.94 ± 0.19 μM, respectively. Similarly, IA-2 has no ability to inhibit the release of elastase from human neutrophils activated by MMK1 (Figure 4C), indicating that IA-2 is selective in inhibiting the release of elastase from human neutrophils, and can specifically inhibit the release of elastase caused by activation of cells via the FPR1 (but not FPR2) signaling pathway. In addition, IA-2 also has different degrees of ability to inhibit the release of elastase caused by the activation of three other factors that can activate human neutrophils, including IL-8, LTB4, and NaF (Figures 4D-4F). In summary, IA-2 does have the effect of inhibiting the release of elastase by activated human neutrophils, and therefore has anti-inflammatory effects.

2.3 IA-1 and IA-2 Effects on superoxide anion generation

This experiment investigated the ability of IA-1 and IA-2 to inhibit the production of superoxide anions by human neutrophils. The relevant experimental results are shown in Figures 5A-5E (for IA-1) and Figures 6A-6E (for IA-2).

As shown in Figures 5A-5B, IA-1 can inhibit the production of superoxide anions by human neutrophils activated by fMLF or fMMYALF, with IC ₅₀ values of 0.81 ± 0.14 μM and 0.52 ± 0.05 μM, respectively. However, IA-1 has no ability to inhibit the production of superoxide anions by human neutrophils activated by MMK1 (Figure 5C), indicating that IA-1 is selective in inhibiting the production of superoxide anions by human neutrophils, and can specifically inhibit the production of superoxide anions caused by activation of cells via the FPR1 (but not FPR2) signaling pathway. In addition, this experiment also tested two other factors that can induce human neutrophils to release superoxide anions, including PMA and NaF. The experimental results are shown in Figures 5D-5E. IA-1 also has different degrees of ability to inhibit the generation of superoxide anions. In summary, IA-1 does have the effect of inhibiting the generation of superoxide anions by activated human neutrophils, and therefore has anti-inflammatory effects.

Similar experimental results can also be seen in the experimental results of IA-2 inhibiting the production of superoxide anions by activated human neutrophils. As shown in Figures 6A-6B, IA-2 can inhibit the production of superoxide anions by human neutrophils activated by fMLF or fMMYALF, and its IC ₅₀ is 0.72 ± 0.07 μM and 0.52 ± 0.07 μM, respectively. Similarly, IA-2 has no ability to inhibit the production of superoxide anions by human neutrophils activated by MMK1 (Figure 6C), indicating that IA-2 is selective in inhibiting the production of superoxide anions by human neutrophils, and can specifically inhibit the production of superoxide anions caused by the activation of cells by the FPR1 (but not the FPR2) signaling pathway. In addition, IA-2 also has different degrees of ability to inhibit the generation of superoxide anions induced by PMA and NaF (Figures 4D-4E). In summary, IA-2 does have the effect of inhibiting the generation of superoxide anions by activated human neutrophils, and therefore has anti-inflammatory effects.

The results of the IC ₅₀ experiments on the effects of IA-1 and IA-2 on inhibiting superoxide anion production and elastin release from human neutrophils are summarized in Tables 2 and 3.

Table 2 IC ₅₀ of IA-1 for superoxide anion generation and elastin release Agonist Superoxide anion generation Flexase release IC ₅₀ (μM) ^a IC ₅₀ (μM) ^a f 0.81 ± 0.14 0.72 ± 0.11 f 0.52 ± 0.05 0.84 ± 0.30 MMK1 > 10 > 10 NaF > 10 > 10 PMA > 10 ND IL-8 ND 3.36 ± 0.46 LTB ₄ ND > 10 Note 1: ^a The concentration required to achieve half-inhibitory concentration (IC ₅₀ ). Note 2: ND: Not detectable concentration was reached.

Table 3 IC ₅₀ of IA-2 for superoxide anion generation and elastin release Agonist Superoxide anion generation Flexase release IC ₅₀ (μM) ^a IC ₅₀ (μM) ^a f 0.72 ± 0.07 0.85 ± 0.28 f 0.52 ± 0.07 0.94 ± 0.19 MMK1 > 10 > 10 NaF > 10 > 10 PMA > 10 ND IL-8 ND > 10 LTB ₄ ND > 10 Note 1: ^a The concentration required to achieve half inhibitory concentration (IC ₅₀ ). Note 2: ND: Not detectable concentration was reached.

2.4 IA-1 and IA-2 For FPR1 Binding ability

Since IA-1 and IA-2 can inhibit the activation of human neutrophils induced by exogenous FPR1 agonist fMLF or endogenous FPR1 agonist fMMYALF, it is speculated that IA-1 and IA-2 inhibit the activation of human neutrophils through the signal transduction pathway of FPR1. In order to understand the mechanism of action of IA-1 and IA-2, it was tested whether IA-1 and IA-2 would bind to FPR1. The relevant experimental results are shown in Figures 7A-7B, which respectively show the binding ability of IA-1 and IA-2 to FPR1. The results in Figures 7A-7B show that IA-1 and IA-2 (0-5 μM) significantly inhibit the binding of another FPR1-specific ligand (fNLFNYK) to FPR1 in a dose-dependent manner, which means that IA-1 and IA-2 inhibit the activation of human neutrophils by blocking a series of signal transduction pathways related to FPR1 through binding to FPR1.

2.5 IA-1 and IA-2 For FPR1 Effects on human neutrophil activation

In order to further verify that IA-1 and IA-2 indeed inhibit the activation of human neutrophils through the signal transduction pathway of FPR1, the following experiments were conducted to prove this. The relevant experimental results are shown in Figures 8A-8B (for IA-1) and 9A-9B (for IA-2). The results in Figures 8A-8B show that as the concentration of fMLF increases, the ability of human neutrophils activated by fMLF to release elastase (Figure 8A) or generate superoxide anions (Figure 8B) increases, that is, a fixed concentration of IA-1 (1 or 2.5 μM) exhibits a decreasing inhibitory effect on the activation of fMLF. Similarly, as shown in Figures 9A-9B, the release of elastase (Figure 9A) or generation of superoxide anions (Figure 9B) by human neutrophils activated by fMLF also increased with the increase in fMLF concentration, and a fixed concentration of IA-2 (1 or 2.5 μM) also showed a decreasing inhibitory effect on fMLF activation. The above results confirm that IA-1 and IA-2 can indeed inhibit the activation of human neutrophils induced by fMLF (via the FPR1 signaling pathway).

2.6 IA-1 and IA-2 For CD11b Impact of performance

Since activated human neutrophils express a surface adhesion factor CD11b to regulate the cell's own adhesion and migration to participate in inflammatory responses, the purpose of this experiment is to confirm the ability of IA-1 and IA-2 to inhibit the expression of CD11b by human neutrophils. The relevant experimental results are shown in Figures 10A-10C (for IA-1) and Figures 11A-11C (for IA-2).

As shown in Figures 10A-10C, IA-1 can inhibit the expression of CD11b by human neutrophils activated by fMLF or fMMYALF (Figures 10A-10B), but cannot inhibit the expression of CD11b by human neutrophils activated by MMK1 (Figure 10C), indicating that IA-1 specifically inhibits the expression of CD11b caused by cell activation via the FPR1 (but not FPR2) signaling pathway. Similarly, as shown in Figures 11A-11C, IA-2 can inhibit the expression of CD11b by human neutrophils activated by fMLF or fMMYALF (Figures 11A-11B), but cannot inhibit the expression of CD11b by human neutrophils activated by MMK1 (Figure 11C). This shows that IA-2 also specifically inhibits the expression of CD11b caused by cell activation via the FPR1 (rather than FPR2) signaling pathway. In summary, IA-1 and IA-2 do have the function of inhibiting the expression of CD11b by activated human neutrophils, thereby inhibiting the participation of activated human neutrophils in inflammatory responses, and therefore have anti-inflammatory effects.

2.7 IA-1 and IA-2 Effects on intracellular calcium release

Since activated human neutrophils increase intracellular calcium ion concentration to promote more human neutrophils to be activated and participate in inflammatory response, this experiment aims to confirm the ability of IA-1 and IA-2 to inhibit the release of calcium ions from human neutrophils. The relevant experimental results are shown in Figure 12 (for IA-1) and Figure 13 (for IA-2).

As shown in Figures 12-13, IA-1 and IA-2 (5 μM) can inhibit the release of calcium ions from human neutrophils activated by fMLF (0.1 μM; left column of Figures 12-13) or fMMYALF (0.1 μM; right column of Figures 12-13), respectively. The specific experimental data are provided in Tables 4 and 5, respectively. Based on the above results, it has been clearly confirmed that IA-1 and IA-2 do have the effect of inhibiting the release of calcium ions from activated human neutrophils, thereby inhibiting the participation of activated human neutrophils in inflammatory reactions, and therefore have anti-inflammatory effects.

Table 4 Experimental data of IA-1 in measuring intracellular calcium ion release Drugs Peak [Ca ²⁺ ] _i (nM) Significance t _1/2 (seconds) Significance 0.1 μM fMLF 303.00 ± 11.61 25.14 ± 3.56 5 μM IA-1 + 0.1 μM fMLF 149.31 ± 16.48 *** 4.52 ± 1.28 *** 0.1 μM fMMYALF 290.71 ± 10.72 21.06 ± 3.00 5 μM IA-1 + 0.1 μM fMMYALF 73.03 ± 8.03 *** 6.00 ± 0.73 ** Note: ** P ＜ 0.01; *** P ＜ 0.001, compared with the control group.

Table 5 Experimental data of IA-2 in measuring intracellular calcium ion release Drugs Peak [Ca ²⁺ ] _i (nM) Significance t _1/2 (seconds) Significance 0.1 μM fMLF 304.37 ± 10.58 24.42 ± 7.05 5 μM IA-2 + 0.1 μM fMLF 187.67 ± 12.66 *** 4.86 ± 0.56 *** 0.1 μM fMMYALF 293.14 ± 5.39 25.14 ± 2.25 5 μM IA-2 + 0.1 μM fMMYALF 97.18 ± 10.05 *** 5.04 ± 0.41 *** Note: *** P ＜ 0.001, compared with the control group.

2.8 IA-1 For treatment ARDS Benefits

From the above experimental results, it can be seen that IA-1 and IA-2 have significant inhibitory effects on human neutrophil activation. Therefore, IA-1 was used as an example for further discussion, confirming that IA-1 does have therapeutic effects on ARDS in the living animal model. The relevant experimental results are shown in Figures 14A-14D. As shown in the immunohistochemical analysis results in Figure 14A, administration of LPS to mice alone will cause a large number of human neutrophils to accumulate in the lung tissue (regardless of the number of nuclear stained cells in H&E staining (Figure 14B) or those with anti-Ly6g reactions (Figure 14C)). However, administration of IA-1 to mice before LPS stimulation can prevent a large number of human neutrophils from infiltrating the lung tissue. At the same time, because the number of human neutrophils in the lung tissue is reduced, the amount of elastase released can be greatly reduced (Figure 14D), thereby protecting the lung tissue from damage. The corresponding staining area statistical analysis results are shown in Figures 14B-14D. The above experimental results clearly demonstrate that IA-1 has the ability to effectively prevent and treat ARDS in mice.

In summary, the experimental data of the present disclosure clearly demonstrate that the present disclosure, a biologically active extract extracted from a culture medium of marine Bacillus sporogenes, and the active ingredients IA-1 and IA-2 separated therefrom can indeed achieve significant inhibition of human neutrophil activation by blocking the signal transduction pathway of FPR1, and also exhibit significant therapeutic efficacy in treating ARDS in mice. Based on the above results, the present disclosure provides a novel drug that can act through FPR1 for safe and effective treatment and/or prevention of ARDS.

Although the above embodiments disclose specific embodiments of the present invention, they are not intended to limit the present invention. A person having ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the principle and spirit of the present invention. Therefore, the scope of protection of the present invention shall be based on that defined by the scope of the attached patent application.

without

In order to make the above and other purposes, features, advantages and embodiments of the present invention more clearly understood, the attached drawings are described as follows:

FIG. 1 is a result obtained according to an embodiment of the present disclosure, illustrating the ability of the eluted fraction (labeled as IALB-E) obtained from a culture medium of a marine bacillus extract after purification by preparative high performance liquid chromatography (PHPLC) to block the binding of N -formyl-Nle-Leu-Phe-Nle-Tyr-Lys ( fNLFNYK ; FPR1 ligand) to its receptor FPR1, wherein N -formyl-L-methionyl-L-leucine-L-phenylalanine- N -form ... -formyl-methionyl-leucyl-phenylalanine, fMLF; exogenous FPR1 agonist, analog of fNLFNYK) as positive control group; micromolar concentration (μM); nanomolar concentration (nM);

Figures 2A-2D are the results of mouse experiments obtained according to an embodiment of the present disclosure, illustrating the effect of IALB-E on inhibiting the release of elastase induced by lipopolysaccharide (LPS) in the lungs of mice, wherein Figure 2A is an immunohistochemical analysis result diagram; Figures 2B-2D are statistical analysis results of Figure 2A, wherein Figure 2B is the statistical analysis result of hematoxylin-eosin (H&E) staining area, and Figure 2C is the statistical analysis result of Ly6g (lymphocyte antigen 6 complex locus G6D (lymphocyte antigen 6 complex locus Figure 2D) is the statistical analysis result of the staining area of a human neutrophil marker, and Figure 2D is the statistical analysis result of the elastin staining area; pre-treatment: IALB-E was administered before LPS administration; post-treatment: IALB-E was administered after LPS administration;

Figures 3A-3F are the results obtained according to an embodiment of the present disclosure, illustrating the effect of the compound anteiso-C13-surfactin (labeled as IA-1) on the release of elastase induced by stimulation of human neutrophils with specific agonists, wherein the specific agonists are: fMLF (exogenous FPR1 agonist; Figure 3A); formyl-Met-Met-Tyr-Ala-Leu-Phe, fMMYALF; endogenous FPR1 agonist; Figure 3B); MMK1 (FPR2 agonist; Figure 3C); IL-8 (CXCR1/2 agonist; Figure 3D); LTB ₄ (TLR agonist; Figure 3E); sodium fluoride (NaF, G protein agonist; Figure 3F); millimolar concentration (mM);

Figures 4A-4F are the results obtained according to an embodiment of the present disclosure, illustrating the effect of the compound iso-C13-surfactin (labeled as IA-2) on the release of elastic enzymes induced by stimulation of human neutrophils with specific agonists, wherein the specific agonists are: fMLF (Figure 4A); fMMYALF (Figure 4B); MMK1 (Figure 4C); IL-8 (Figure 4D); LTB ₄ (Figure 4E); NaF (Figure 4F);

Figures 5A-5E are the results obtained according to an embodiment of the present disclosure, illustrating the effect of compound IA-1 on the generation of superoxide anions induced by stimulation of human neutrophils with specific agonists, wherein the specific agonists are: fMLF (Figure 5A); fMMYALF (Figure 5B); MMK1 (Figure 5C); phorbol myristate acetate (PMA; protein kinase C (PKC) activator; Figure 5D); NaF (Figure 5E);

Figures 6A-6E are the results obtained according to an embodiment of the present disclosure, illustrating the effect of compound IA-2 on the generation of superoxide anions induced by stimulation of human neutrophils with specific agonists, wherein the specific agonists are: fMLF (Figure 6A); fMMYALF (Figure 6B); MMK1 (Figure 6C); PMA (Figure 6D); NaF (Figure 6E);

Figures 7A-7B are the results obtained according to an embodiment of the present disclosure, illustrating the ability of IA-1 (Figure 7A) or IA-2 (Figure 7B) to block the binding of fNLFNYK to its receptor FPR1, wherein fMLF is a positive control group;

Figures 8A-8B are the results obtained according to an embodiment of the present disclosure, illustrating the effect of compound IA-1 on inhibiting the release of elastic enzymes (Figure 8A) or superoxide anion generation (Figure 8B) induced by stimulation of human neutrophils with fMLF at a specific concentration;

Figures 9A-9B are the results obtained according to an embodiment of the present disclosure, illustrating the effect of compound IA-2 on inhibiting the release of elastic enzymes (Figure 9A) or superoxide anion generation (Figure 9B) induced by stimulation of human neutrophils with fMLF at a specific concentration;

Figures 10A-10C are the results obtained according to an embodiment of the present disclosure, illustrating the effect of compound IA-1 on inhibiting the expression of surface adhesion factor CD11b induced by stimulation of human neutrophils with specific agonists, wherein the specific agonists are: fMLF (Figure 10A); fMMYALF (Figure 10B); MMK1 (Figure 10C);

Figures 11A-11C are the results obtained according to an embodiment of the present disclosure, illustrating the effect of compound IA-2 on inhibiting the expression of CD11b induced by stimulation of human neutrophils with specific agonists, wherein the specific agonists are: fMLF (Figure 11A); fMMYALF (Figure 11B); MMK1 (Figure 11C);

FIG. 12 is a result obtained according to an embodiment of the present disclosure, illustrating the effect of compound IA-1 on inhibiting the increase in intracellular calcium ion (Ca ²⁺ ) concentration induced by stimulation of human neutrophils with specific agonists, wherein the specific agonists are: fMLF (left column); fMMYALF (right column);

FIG. 13 is a result obtained according to an embodiment of the present disclosure, illustrating the effect of compound IA-2 on inhibiting the increase in intracellular calcium ion concentration induced by stimulation of human neutrophils with specific agonists, wherein the specific agonists are: fMLF (left column); fMMYALF (right column); and

Figures 14A-14D are the results of mouse experiments obtained according to an embodiment of the present disclosure, illustrating the effect of IA-1 on inhibiting the release of elastin induced by lipopolysaccharide stimulation in the mouse lungs, wherein Figure 14A is an immunohistochemical analysis result diagram; Figures 14B-14D are the statistical analysis results of Figure 14A, wherein Figure 14B is the statistical analysis result of H&E staining area, Figure 14C is the statistical analysis result of Ly6g staining area, and Figure 14D is the statistical analysis result of elastin staining area.

Claims (5)

A use of a compound of formula (Ib) or a pharmaceutically acceptable salt thereof in the preparation of a medicament, wherein the medicament is administered in an effective amount to treat and/or prevent acute respiratory distress syndrome (ARDS) in a subject:

The use as described in claim 1, wherein the compound of formula (I-b) or a pharmaceutically acceptable salt thereof is a type I formyl peptide receptor (FPR1) antagonist. The use as claimed in claim 1, wherein the compound of formula (Ib) is extracted from a biologically active secondary metabolite of a marine Bacillus sp. The use as described in claim 1, wherein the effective amount is 0.1-10 mg/kg. The use as described in claim 1, wherein the individual is a human.

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