KR102570075B1 - Pharmaceutical composition for preventing or treating Mycobacterium species infection and method using the same - Google Patents

Abstract

Compounds containing the peptide of formula 1, isomers, derivatives, solvates, or pharmaceutically acceptable salts thereof, for preventing or treating bacterial infections of Mycobacterium sp. or symptoms associated therewith. A composition and a method using the same are provided. According to this, it has anti-tuberculosis activity against mycobacterium, for example, Mycobacterium tuberculosis, and can alleviate the inflammatory reaction caused by Mycobacterium tuberculosis, thereby preventing or preventing bacterial infections of the genus Mycobacterium or symptoms associated therewith. can be used for treatment.

Description

Pharmaceutical composition for preventing or treating bacterial infection of the genus Mycobacterium including cyclic peptide compound and method using the same

It relates to a pharmaceutical composition for preventing or treating bacterial infection of the genus Mycobacterium including a cyclic peptide compound and a method using the same.

Tuberculosis is an infectious disease that has existed for such a long time that its traces were found even in the Stone Age around 7,000 BC. Despite being an old disease, it still accounts for a significant number of deaths among young people worldwide. Mycobacterium tuberculosis, which causes tuberculosis, was discovered in 1882 by the German bacteriologist Robert Koch. Tuberculosis does not occur in all patients who are infected with Mycobacterium tuberculosis. Currently, it is estimated that about 30% of the world's population is infected with Mycobacterium tuberculosis. Although Mycobacterium tuberculosis has a slower growth rate than other infectious bacteria, it is characterized by resistance to various drugs by causing mutations while surviving in macrophages. Therefore, as a tuberculosis treatment method, it is difficult to see a therapeutic effect with a general single drug prescription, and long-term treatment with a combination of various drugs is recommended.

Various compounds such as isoniazid, rifampicin, ethambutol, pyrazinamide, streptomycin, and kanamycin are used as anti-tuberculosis agents. However, isoniazid can cause peripheral neuritis, rifampicin causes thrombocytopenia, streptomycin causes balance disorder or hearing loss, or peripheral neuropathy, ethambutol can cause visual impairment, and pyrazinamide causes high uric acid It can also cause blood loss. As such, there is a problem in that the types of anti-tuberculosis drugs are not diverse and the number of prescriptions or dose is limited due to various side effects of each drug. In addition, compounds having anti-tuberculosis activity are being developed (Korean Publication No. 10-2015-0144812 (2015.12.28)).

Therefore, it is necessary to select a new antituberculous agent having antituberculous activity and fewer side effects.

Provided is a pharmaceutical composition for preventing or treating bacterial infections of Mycobacterium sp. or symptoms associated therewith, comprising a cyclic peptide compound, an isomer, derivative, solvate, or pharmaceutically acceptable salt thereof. .

Provided is a method for preventing or treating a bacterial infection of the genus Mycobacterium or symptoms associated therewith using a cyclic peptide compound, an isomer, derivative, solvate, or pharmaceutically acceptable salt thereof.

According to one aspect, a compound containing the peptide of Formula 1, an isomer, derivative, solvate, or pharmaceutically acceptable salt thereof for the prevention or treatment of a bacterial infection of the genus Mycobacterium or symptoms associated therewith. composition is provided.

According to another aspect, a method for preventing or treating a bacterial infection of the genus Mycobacterium or symptoms associated therewith using a pharmaceutical composition comprising the compound, its isomer, derivative, solvate, or pharmaceutically acceptable salt thereof to provide.

Compounds comprising the peptide of formula 1, isomers, derivatives, solvates, or pharmaceutically acceptable salts thereof have anti-tuberculosis activity against mycobacteria, for example Mycobacterium tuberculosis, and are caused by Mycobacterium tuberculosis. Since it can alleviate the inflammatory response, it can be used to prevent or treat bacterial infections of the genus Mycobacterium or symptoms related thereto.

1a and 1b are structural formulas of stigmata amycin A (OMS-A) and stomatamicin B (OMS-B), respectively.
Figure 2 is a graph showing anti-tuberculosis activity according to the concentration of the compound (○: OMS-A, ◆: OMS-B, □: isoniazid, ●: ethambutol, ■: SQ109, RFU: relative fluorescence unit )).
3A and 3B are graphs showing colony forming units (CFU) according to culture time (days) upon administration of OMS-A and OMS-B, respectively.
4 is a graph showing CFU values according to concentrations of OMS-A, OMS-B, or a combination thereof.
5A and 5B are graphs showing the survival rate (%) of Drosophila infected with Mycobacterium marinum and the CFU value of M. marinum present in Drosophila according to the concentration of OMS-A, respectively.
Figure 6a is an image of macrophages stained with DAPI and anti-LC3 antibody in the presence of OMS-A or OMS-B, and Figure 6b is the number of LC3 spots per macrophage in the presence of OMS-A or OMS-B is a graph that represents
Figure 7a is an immunoblotting image of macrophages according to the presence of OMS-A or OMS-B, 3-methyladenine (3-MA) or bafilomycin A1 (Bafilomycin A1: Baf-A) It is an immunoblotting image of macrophages according to pre-treatment.
8 is a graph showing the results of flow cytometry analysis of macrophages treated with OMS-A or OMS-B.
9 is an image of immunofluorescence staining of macrophages treated with OMS-A or OMS-B.
Figures 10a and 10b are graphs showing the ratio (%) of the common region between phagosomes and LC3, respectively, fluorescence images obtained for LC3 and Mtb, and Figures 10c and 10d are fluorescence images obtained for LAMP2 and Mtb, respectively. It is a graph showing the ratio (%) of the co-region of phagosome and LAMP2.
11a and 11b are immunoblotting images obtained by OMS-A and OMS-B, respectively.
Figure 12a is a graph showing the amount of cytokines secreted from Mycobacterium tuberculosis-infected macrophages according to the amount of Omyeongsamycin, and Figure 12b is a graph showing the amount of cytokines secreted from Mycobacterium tuberculosis-infected macrophages when OMS-A or OMS-B was treated, NF- It is a graph showing the expression level of κB.

One aspect is a compound comprising the peptide of formula 1, its isomers, derivatives, solvates, or pharmaceutically acceptable salts thereof, including Mycobacterium sp. Prevention of bacterial infections or symptoms associated therewith, or A pharmaceutical composition for treatment is provided.

[Equation 1]

.

In Formula 1, Val, Thr, Leu, Trp, and Phe respectively represent valine, threonine, leucine, tryptophan, and phenylalanine.

In the above formula, R is H, a substituted or unsubstituted C ₁ to C ₂₀ alkyl group, a substituted or unsubstituted C ₂ to C ₂₀ alkenyl group, a substituted or unsubstituted C 2 to C 20 alkanyl group, or a substituted or unsubstituted C ₂ to C ₂₀ alkenyl group. It may be a C ₃ to C ₂₀ aryl group, or a combination thereof. The alkyl group may be, for example, a C ₁ to C ₁₅ , C ₁ to C ₁₀ , C ₁ to C ₅ , or C ₁ to C ₃ alkyl group. The alkyl group may be, for example, a methyl group. The alkenyl group may be, for example, an alkenyl group of C ₂ to C ₁₅ , C ₂ to C ₁₀ , or C ₂ to C ₅ . The alkanyl group may be, for example, a C ₂ to C ₁₅ , C ₂ to C ₁₀ , or C ₂ to C ₅ alkanyl group. The aryl group may be, for example, an aryl group of C ₃ to C ₁₅ , C ₃ to C ₁₀ , or C ₃ to C ₅ .

Amino acids in the compounds may be substituted with other amino acids having similar physicochemical properties. Valine, leucine, tryptophan, or phenylalanine may be substituted with other amino acids or derivatives thereof having a non-polar R group. For example, valine may be substituted with alanine (Ala), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tryptophan (Trp) or methionine (Met). For example, leucine may be substituted with alanine (Ala), valine (Val), isoleucine (Ile), phenylalanine (Phe), tryptophan (Trp) or methionine (Met). For example, tryptophan may be substituted with alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), or methionine (Met). For example, phenylalanine may be substituted with alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), tryptophan (Trp) or methionine (Met). Threonine may be substituted with other amino acids or derivatives thereof having a polar R group. For example, threonine may be substituted by substitution with glycine (Gly), serine (Ser), cysteine (Cys), tyrosine (Tyr), asparagine (Asn) or glutamine (Gln).

The Trp may be 4-OCH ₃ -Trp, in which a methoxy group is bonded to the 4th carbon of the R group.

The Phe may be β-OH-Phe in which a hydroxyl group is bonded to the β carbon.

The compound may be a compound in which a C ₁ to C ₂₀ , C ₁ to C ₁₅ , C ₁ to C ₁₀ , C ₁ to C ₅ , or C ₁ to C ₃ alkyl group is bonded to an amino group of an amino acid. For example, Trp, Leu, Val ¹ , Val ⁵ , Thr ² , or a combination of these amino acids may be a compound in which a C ₁ to C ₂₀ alkyl group is bonded to an amino group thereof. For example, the alkyl group may be methyl.

The compound may be a compound represented by Formula 2.

[Equation 2]

.

In Formula 2, R is H, a substituted or unsubstituted C ₁ to C ₂₀ alkyl group, a substituted or unsubstituted C 2 to C ₂₀ alkenyl group, a substituted or unsubstituted C ₂ to C 20 alkanyl group, or a substituted or unsubstituted C ₂ to C ₂₀ alkanyl group. It may be a cyclic C ₃ to C ₂₀ aryl group, or a combination thereof.

The compound may be a compound represented by Formula 3.

[Equation 3]

.

The compound may be a compound represented by Formula 4.

[Equation 4]

.

The above "substitution" refers to introduction in place of a hydrogen atom when a derivative is formed by substituting one or more hydrogen atoms in an organic compound with another atomic group, and "substituent" refers to an introduced atomic group.

Substituents include, for example, a halogen atom, a halogen atom, a C ₁ to C ₂₀ alkyl group substituted with a halogen atom (eg, CCF ₃ , CHCF ₂ , CH ₂ F, CCl _{3 ,} etc.), a C ₁ to C ₂₀ alkoxy, C ₂ to _C20 alkoxyalkyl, hydroxy group, nitro group, cyano group, amino group, amidino group, hydrazine, hydrazone, carboxyl group or its salt, sulfonyl group, sulfamoyl group, sulfonic acid group or its salt, phosphoric acid or its salt A salt, or a C ₁ to C ₂₀ alkyl group, a C ₂ to C ₂₀ alkenyl group, a C ₂ to C ₂₀ alkynyl group, a C ₁ to C ₂₀ heteroalkyl group, a C ₆ to C ₂₀ aryl group, a C ₆ to C ₂₀ arylalkyl group, It may be a C ₆ to C ₂₀ heteroaryl group, a C ₇ to C ₂₀ heteroarylalkyl group, a C ₆ to C ₂₀ heteroaryloxy group, and a C ₆ to C ₂₀ heteroaryloxyalkyl group or a C ₆ to C ₂₀ heteroarylalkyl group. .

The term "alkyl" refers to a fully saturated branched or unbranched (or straight chain or linear) hydrocarbon. The alkyl is C ₁ to C ₂₀ , C ₁ to C ₁₅ , C ₁ to C ₁₀ , or C ₁ to C ₅ alkyl group. Non-limiting examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, neopentyl, iso-amyl, n-hexyl, 3-methyl hexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, or n-heptyl; and the like.

The term "alkenyl" refers to a branched or unbranched hydrocarbon having at least one carbon-carbon double bond. The C ₂ -C ₂₀ alkenyl group may be, for example, a C ₂ to C ₁₅ , C ₂ to C ₁₀ , or C ₂ to C ₅ alkenyl group. Non-limiting examples of alkenyl groups include vinyl, allyl, butenyl, isopropenyl, or isobutenyl.

The term "alkynyl" refers to a branched or unbranched hydrocarbon having at least one carbon-carbon triple bond. The C ₂ -C ₂₀ alkynyl group may be, for example, a C ₂ to C ₁₅ , C ₂ to C ₁₀ , or C ₂ to C ₅ alkynyl group. Non-limiting examples of alkynyl include ethynyl, butynyl, isobutynyl, isopropynyl, and the like.

The term “aryl” also includes groups in which an aromatic ring is fused to one or more carbon rings. The C ₆ -C ₃₀ aryl group may be, for example, a C ₆ to C ₁₅ or C ₆ to C ₁₀ aryl group. Non-limiting examples of aryl include phenyl, naphthyl, or tetrahydronaphthyl, and the like.

The term "isomer" refers to a compound having the same molecular formula but not the same connection method or spatial arrangement of constituent atoms in a bonsai. Isomers include, for example, structural isomers, and stereoisomers. The stereoisomers may be diastereomers or enantiomers. Enantiomers refer to isomers that do not overlap with their mirror images, such as the relationship between left and right hands, and are also called optical isomers. Enantiomers are classified as R (Rectus: clockwise) and S (sinister: counterclockwise) when four or more substituents on the chiral central carbon differ from each other. Diastereoisomers are stereoisomers that are not enantiomers, and can be divided into cis-trans isomers that arise from differences in the arrangement of atoms in space.

The term "derivative" refers to a compound obtained by substituting a part of the structure of the compound with another atom or group of atoms.

The term “solvate” refers to a compound solvated in an organic or inorganic solvent. The solvate is, for example, a hydrate.

The term "pharmaceutically acceptable salt" refers to inorganic and organic acid addition salts of a compound. The pharmaceutically acceptable salt may be a salt that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. The inorganic acid salt may be a hydrochloride, bromate, phosphate, sulfate, or bisulfate salt. The organic acid salt is formate, acetate, acetate, propionate, lactate, oxalate, tartrate, malate, maleate, citrate, fumarate, besylate, camsylate, edisyl salt, trichloroacetic acid, trifluoro Acetate, benzoate, gluconate, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, galacturonate, embonate, glutamate, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or aspartate. The metal salt may be a calcium salt, sodium salt, magnesium salt, strontium salt, or potassium salt.

The bacterial infection of the genus Mycobacterium may be selected from the group consisting of tuberculosis, leprosy, and nontuberculous mycobacteria (NTM) infection. The tuberculosis may be multi drug resistance (MDR) or extensive drug resistance (XDR) tuberculosis. The multidrug-resistant tuberculosis may be tuberculosis in which tuberculosis bacteria are not killed even when isoniazid and rifampicin are administered to the human body. Extensive drug-resistant tuberculosis can be tuberculosis that is simultaneously resistant to isoniazid and rifampicin as well as to quinolones and injections.

Symptoms associated with bacterial infection of the genus Mycobacterium may include cough, chest pain, fever, chills, loss of appetite, weight loss, inflammation, or a combination thereof. For example, symptoms associated with a bacterial infection of the genus Mycobacterium may be inflammation induced by bacteria of the genus Mycobacterium.

The bacteria of the genus Mycobacterium are bacteria belonging to Corynebacterium (Corynebacterineae) of Actinobacteria. Bacteria of the genus Mycobacterium may have acid-fast properties. Bacteria of the genus Mycobacterium include, for example, Mycobacterium tuberculosis, Mycobacterium bovis (M. bovis), Mycobacterium afrakanum (M. africanum), Mycobacterium microti (M. microti), and M. leprae.

A compound comprising the peptide of Formula 1, an isomer, a derivative, or a pharmaceutically acceptable salt thereof may promote autophagy in macrophages, relieve inflammation, or induce a combination thereof.

The composition may further contain an antibiotic. The antibiotic may be an antituberculous drug. The anti-tuberculosis agent is, for example, isoniazid, rifampicin, ethambutol, SQ-109, pyrazinamide, streptomycin, kanamycin, capreomycin , ethionamide, prothionamide, enviomycin, para-aminosalicylic acid, cycloserine, amikacin, levofloxacin ), moxifloxacin, gatifloxacin, ofloxacin, terizidone, thionamide, ethionamide, protionamide, clo phagemine, linezolid, amoxicillin, clavulanate, thioacetazone, imipenem, cilastatin, clarithromycin ), bedaquiline, delamanid, lmipenem, cilastatin, meropenem, or combinations thereof. A compound comprising the peptide of Formula 1, an isomer, derivative, solvate, or pharmaceutically acceptable salt thereof and the antibiotic may be a single or separate composition.

The term "prevention" refers to any action that suppresses or delays the onset of a disease by administration of a composition. The term "treatment" refers to all activities that improve or beneficially change symptoms of a disease by administration of a composition.

The pharmaceutical composition may further include a carrier, excipient or diluent. Carriers, excipients and diluents include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, crude quality cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

The pharmaceutical composition may be formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods. When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

In the pharmaceutical composition, the solid preparation for oral administration may be tablets, pills, powders, granules, or capsules. The solid formulation may further include an excipient. The excipient may be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. In addition, the solid preparation may further include a lubricant such as magnesium stearate or talc. In the pharmaceutical composition, the oral liquid formulation may be a suspension, internal solution, emulsion, or syrup. The liquid formulation may include water or liquid paraffin. The liquid formulation may contain excipients such as wetting agents, sweetening agents, flavoring agents, or preservatives. In the pharmaceutical composition, preparations for parenteral administration may be sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried or suppositories. Non-aqueous solvents or suspensions may include vegetable oils or esters. The vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil. The ester may be, for example, ethyl oleate. The base of the suppository may be witepsol, macrogol, tween 61, cacao butter, laurin fat, or glycerogelatin.

A preferred dosage of the pharmaceutical composition varies depending on the condition and body weight of the subject, the severity of the disease, the type of drug, the route and period of administration, but can be appropriately selected by those skilled in the art. However, the compound, its isomer, derivative, solvate, or pharmaceutically acceptable salt can be used at, for example, about 0.0001 mg/kg to about 100 mg/kg, or about 0.001 mg/kg to about 100 mg/kg. The amount may be divided into 1 to 24 times per day, 1 to 7 times per 2 days to 1 week, or 1 to 24 times per month to 12 months. In the pharmaceutical composition, the compound, its isomer, derivative, solvate, or pharmaceutically acceptable salt is about 0.0001% to about 10% by weight, or about 0.001% to about 1% by weight based on the total weight of the composition. can be included

The administration method may be oral or parenteral administration. The method of administration can be, for example, oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, intranasal, intratracheal, or intradermal routes. The composition may be administered systemically or topically, and may be administered alone or in combination with other pharmaceutically active compounds.

Another aspect provides a method for preventing or treating a bacterial infection of the genus Mycobacterium or symptoms associated therewith, comprising administering the pharmaceutical composition according to one aspect to a subject.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat or cat. The subject may be an individual suffering from, or highly likely to suffer from, a bacterial infection of the genus Mycobacterium or symptoms associated therewith.

The method may further include administering an antibiotic to the subject. The antibiotic may be administered to the subject simultaneously, separately or sequentially with a compound comprising the peptide of Formula 1, an isomer, derivative, or pharmaceutically acceptable salt thereof.

The administration method may be oral or parenteral administration. The method of administration can be, for example, oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, intranasal, intratracheal, or intradermal routes. The pharmaceutical composition may be administered systemically or topically, and may be administered alone or in combination with other pharmaceutically active compounds.

A preferred dosage of the pharmaceutical composition varies depending on the condition and body weight of the patient, the severity of the disease, the type of drug, the route and period of administration, but can be appropriately selected by those skilled in the art. The dosage is, for example, in the range of about 0.001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 10 mg/kg, or about 0.1 mg/kg to about 1 mg/kg on an adult basis. can be The administration may be administered once a day, multiple times a day, or once a week, once every 2 weeks, once every 3 weeks, or once every 4 weeks to once a year.

In this specification, the term "include" is used to indicate that other components may be added or/or intervened, not excluded, unless otherwise stated.

It will be described in more detail through the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example One. of stigma samicin Confirmation of anti-tuberculosis activity

One. Streptomyces genus SNJ042 Cultivation of strains

A marine-derived bacterium Streptomyces genus SNJ042 strain was prepared (Korean Patent Registration No. 10-1522625 (2015.05.18)).

SNJ042 strain of the genus Streptomyces was inoculated into YEME solid medium (malt extract 10 g, yeast extract 4 g, glucose 2 g, artificial sea salt 34 g, agar 18 g per 1 L of water), and the first cultured.

Then, the spores produced by the strain were inoculated into 125 ml of YEME liquid medium (malt extract 10 g, yeast extract 4 g, glucose 4 g, artificial sea salt 34 g per 1 L of water), and about 28 ° C. for about 2 days. It was cultured while shaking at a speed of about 180 rpm at a temperature of. 14 ml of liquid culture was inoculated into 1 L of A1+C liquid medium (10 g of starch, 4 g of enzyme extract, 2 g of peptone, 1 g of calcium carbonate, 34 g of artificial sea salt per 1 L of water), and about 6 to 7 It was cultured while shaking at a speed of about 180 rpm at a temperature of about 28 ° C. for days.

A total of 108 of the above strains were inoculated in a 1 L liquid medium, and the SNJ042 strain of the genus Streptomyces was cultured in a total of 108 L of the medium.

2. of stigma samicin separation

Into the separatory funnel, 1 L of the culture medium of strain SNJ042 cultured as described in 1. and 1.5 L of ethyl acetate were injected. After sealing the inlet of the funnel, mixing the solution vigorously for about 2 minutes and then leaving it for about 1 minute was repeated twice, and left for 3 minutes. After that, the water layer was removed and an ethyl acetate layer was obtained. After adding 100 g of anhydrous sodium sulfate to the obtained ethyl acetate layer to remove moisture, the ethyl acetate layer was filtered through filter paper. The filtered solution was reduced under reduced pressure to evaporate ethyl acetate and the solution was concentrated. This process was repeated to obtain 12 g of crude extract from a total of 108 L of culture.

The crude extract was divided into 5 fractions using reverse phase chromatography (Sepak® C ₁₈ 20 g, reversed-phase) twice. Water and methanol were used as solvents for fractionation, and 20% (v/v), 40% (v/v), 60% (v/v), 80% (v/v), and 100% each of 200 ml % (v/v) methanol/water solution was used. Fractionation was started using a 20% (v/v) methanol/water solution, and then fractionation was performed with increasing concentrations of methanol (20% (v/v) methanol/water → 100% (v/v) methanol). proceeded. After the fractionation using 100% (v/v) methanol was completed, 20 µl of each of the 5 fractions of 200 ml each was transferred to a vial for LC/MS analysis and analyzed using LC/MS. The column used was a reversed-phase HPLC column (Phenomenex, C ₁₈ (2), 100 X 4.6 mm), and the gradient solvent conditions (10% (v / v) to 100% (v / v for 20 minutes) ) in acetonitrile/water). Through the retention time ( t _R ), ultraviolet spectrum, and mass spectrum, the target material was found in fraction 4 (80% (v/v) methanol/water) and fraction 5 (100% (v/v) methanol/water). It was confirmed that Ohmyungsamycin (OMS) -A and OMS-B were included.

Fractions 4 and 5 were concentrated in a vacuum concentrator and dissolved in 100% (v/v) methanol, and the concentration gradient solvent conditions (40% (v/v) to 56% (v/v) acetonitrile/water for 20 minutes) , and then separated by a reverse phase HPLC column (Kromacil, C ₁₈ (2), 250 X 10 mm) using 60% (v/v) acetonitrile/water). OMS-A and OMS-B compounds were eluted at about 32 and 34 minutes, respectively, after HPLC injection under the above separation conditions. The obtained OMS-A and OMS-B were confirmed for purity using LC/MS as described above. Finally, pure 70 mg of OMS-A and 20 mg of OMS-B were isolated.

3. Against Mycobacterium tuberculosis of stigma samicin Determination of the Minimum Inhibitory Concentration

To determine whether OMS-A and OMS-B inhibit Mycobacterium tuberculosis (Mtb), the minimum inhibitory concentration (MIC) of OMS-A and OMS-B was measured.

To measure the MIC of OMS-A and OMS-B, resazurin-based microtiter plate assay (REMA) was performed. An Mtb H37Rv strain (University of Arizona) was prepared, and the prepared Mtb H37Rv strain was prepared through dilution culture so that an OD ₆₀₀ value was 0.005. Each well of a 96-well plate was inoculated with 100 μl of Mtb H37Rv strain culture.

As test compounds, OMS-A and OMS-B prepared as described in 2. were used. As positive controls, isoniazid (INH) (Sigma) and ethambutol (EMB) (Sigma), which are antibiotics used as a tuberculosis treatment, and SQ109 (Sigma) under development as a tuberculosis treatment (Stehr et al., Curr . Top. Med . Chem . , 2014, vol.14, p.110-129) was used. The compounds were diluted by a 2-fold serial dilution method. The prepared compound and 40 μl of 0.025% (w/v) resazurin (Sigma) solution were added to each well, and each plate was incubated at about 37° C. for about 5 days.

On day 1 after starting the culture, the amount of resorufin, a metabolite of resazurin, was measured using a Synergy H1 micro plate reader (BioTek). The MIC ₅₀ (50% minimum inhibitory concentration of bacterial growth) was determined from the amount of resorufin measured using GraphPad Prism 5.0 software. The measured fluorescence intensity (relative fluorescence unit: RFU) according to the concentration of the compound is shown in FIG. 2 .

As shown in Figure 2, the OMS-A and OMS-B compounds have superior anti-tuberculosis activity compared to isoniazid, ethambutol, and SQ109, and the MIC ₅₀ of the OMS-A and OMS-B compounds are 57 nM and 117 nM, respectively. was

4. Against macrophages infected with Mycobacterium tuberculosis of stigma samicin antibacterial activity

Bone-marrow-derived macrophages (BMDM) collected from wild-type mice (Samtako Bio Korea) were cultured on plates. Mtb H37Rv strain at a multiplicity of infection (MOI) of 10 was added to the cultured macrophages, and incubated for about 4 hours at a temperature of about 37° C. and 5% CO ₂ .

1 μM or 10 μM of OMS-A, 1 μM or 10 μM of OMS-B, or a combination thereof were added to macrophages infected with Mycobacterium tuberculosis, respectively, and cultured for about 3 days at a temperature of about 37° C. and 5% CO ₂ . A solvent control (SC) to which OMS-A and OMS-B were not added was used as a negative control group, and 0.5 μg/ml of isoniazid (INH) (Sigma) was used as a control group. The colony forming unit (CFU) of the cultured macrophages was measured. Upon administration of OMS-A and OMS-B, CFU according to culture time (days) is shown in FIGS. 3a and 3b, respectively. In addition, after administration of the combination of OMS-A and OMS-B and culturing for 3 days, the CFU according to the concentration of the administered compound is shown in FIG. 4 (***: p<0.001).

As shown in Figures 3a and 3b, each of OMS-A and OMS-B had a similar value of CFU to isoniazid and decreased in a concentration-dependent manner. In addition, as shown in FIG. 4, when OMS-A and OMS-B were treated together, the CFU value was lower than that when treated separately. Therefore, it was confirmed that OMS-A and OMS-B have antibacterial activity against macrophages infected with Mycobacterium tuberculosis.

5. In vivo of stigma samicin antibacterial activity

In order to confirm whether stigmata amycin has antibacterial activity in vivo, a Mycobacterium marinum - Drosophila melanogaster infection system was used (Dionne et al., Infect Immun . , 2003, vol.71, p.3540-3550).

Drosophila melanogaster ) was anesthetized by inhalation with CO ₂ gas, and infected by injecting M. marinum adjusted to 500 cfu. Thereafter, Drosophila melanogaster was cultured in a culture medium containing 1 μM or 10 μM of OMS-A. It was transferred to a new culture medium every 3 days, and the survival rate was calculated by measuring the number of Drosophila yellow flies for about 30 days. As a positive control, phosphate-buffered saline (PBS) was added to Drosophila not infected with M. marinum, and Drosophila infected with M. marinum was used as a negative control. Also, in the control group, 1 μg/ml of antibiotic amikacin (AMK) (Sigma) was added to Drosophila infected with M. marinum. The calculated survival rate (%) of Drosophila is shown in FIG. 5a (Mm: Drosophila infected with M. marinum). As shown in Fig. 5a, OMS-A, like amikacin, increased the survival rate of Drosophila infected with M. marinum.

For more accurate results, Drosophila Drosophila was anesthetized, transferred to a new tube, and then homogenized using a homogenizer until only the cuticle layer remained. Thereafter, M. marinum present in Drosophila was extracted and the CFU value was measured as described in 4. The CFU values of M. marinum present in Drosophila according to the concentration of OMS-A are shown in Fig. 5b (***: p<0.001). As shown in Figure 5b, when OMS-A was treated, the CFU value of M. marinum in Drosophila was decreased in a concentration-dependent manner compared to the negative control solvent control (SC).

Therefore, it was confirmed that OMS-A has antibacterial activity not only in vitro but also in vivo.

6. On the activation of macrophage autophagy of stigma samicin effect

It was confirmed whether OMS-A and OMS-B are involved in the activation of autophagy in macrophages.

LC3 puncta formation and lipidation assays, which are mainly used for measuring autophagy of macrophages, were performed (Eisuke and Noboru, Autophagy , 2010, vol.6, p.764-776). 10 μM OMS-A or 10 μM OMS-B was added to bone marrow-derived macrophage (BMDM) prepared as described in 4. and cultured for about 24 hours. PBS was used as a negative control. Macrophages were then stained with DAPI (4',6-diamidino-2-phenylindole) (Sigma) and an anti-LC3 antibody (Cell signaling), and the number of LC3 spots per macrophage was measured. Images of stained macrophages are shown in Fig. 6a (UN: untreated), and the number of LC3 spots per macrophage according to OMS-A or OMS-B is shown in Fig. 6b (**: p<0.01). As shown in FIGS. 6A and 6B , many LC3 spots were formed when OMS-A and OMS-B were treated.

In addition, 1 µM, 5 µM, and 10 µM of OMS-A or OMS-B were added to macrophages, and after culturing for about 24 hours, macrophage cell lysates were obtained. The obtained cell lysate was subjected to immunoblotting using an anti-LC3 antibody (Cell signaling) and an anti-β-actin antibody (Santa Cruz), and the results are shown in FIG. 7a (U: untreated). In addition, 10 µM of 3-methyladenine (3-MA) as an autophagy inhibitor or 100 nM of Bafilomycin A1 (Baf-A) as an H ⁺ -ATPase inhibitor was pre-injected into macrophages. In the case of treatment, immunoblotting images of macrophages are shown in FIG. 7B (U: untreated, SC: solvent control). As shown in FIG. 7a, when OMS-A and OMS-B were treated, the amount of LC2-phosphatidylethanolamine conjugate, LC3-II, increased compared to LC3-I, which is the cytoplasmic form of LC3. In addition, as shown in FIG. 7B, when 3-MA was pre-treated, the amount of LC3-II was reduced compared to when OMS-A or OMS-B was treated separately. When Baf-A was pre-treated, the amount of LC3-II was increased compared to when OMS-A or OMS-B was treated separately.

Meanwhile, 10 µM of OMS-A or OMS-B was added to macrophages and cultured for about 24 hours. Then, the mean fluorescence intensity (MFI) of macrophages was measured by flow cytometry using an anti-LC3B antibody (Cell signaling). The mean fluorescence intensities according to OMS-A and OMS-B are shown in FIG. 8 (*: p<0.01). As shown in Figure 8, it was confirmed that the expression level of LC3B increased in macrophages treated with OMS-A and OMS-B.

Therefore, it was confirmed that OMS-A and OMS-B can enhance autophagy.

In addition, macrophages were transfected with a retroviral vector expressing mCherry-enhanced green fluorescent protein (EGFP)-LC3B. 10 µM of OMS-A or OMS-B was added to the transfected macrophages and cultured for about 24 hours. Fluorescent images of the transfected macrophages were obtained, and the images are shown in FIG. 9 (UN: untreated, N: nuclei). As shown in FIG. 9, when the fluorescence intensity of mCherry-EGFP-LC3B delivered to the lysosome of macrophages was measured, the number of red spots increased. Therefore, it was confirmed that OMS-A and OMS-B enhance not only autophagy but also autophagic flux.

7. Inflammatory response of Mycobacterium tuberculosis induced macrophages of stigma samicin effect

To confirm whether OMS-A and OMS-B can inhibit the maturation of phagolysosomes caused by Mycobacterium tuberculosis (Mtb), macrophages were prepared as described in 4.

Prepared macrophages were transfected with enhanced red fluorescent protein (ERFP)-Mtb (University of Gyeongsang), and then 10 µM of OMS-A or OMS-B was added to the macrophages. After culturing macrophages for about 24 hours, anti-LC3 antibody (MBL), anti-lysosome-associated membrane proein 2 (LAMP2) antibody (Santa Cruz), and DAPI were used to inoculate macrophages. Fluorescent images were obtained. From the obtained fluorescence images, the co-localization ratio (%) of the Mtb phagosome and LC3 or LAMP2 was calculated. The fluorescence image obtained for LC3 and the graph showing the ratio of the common-region of Mtb phagosome and LC3 are shown in FIGS. 10A and 10B, respectively, and the fluorescence image obtained for LAMP2 and the common-region of Mtb phagosome and LAMP2 Graphs showing the ratio of are shown in FIGS. 10c and 10d, respectively (**: p<0.01).

10a to 10d, when OMS-A and OMS-B were treated, co-localization of LC3 and Mtb phagosomes, which are indicators of autophagy, and co-localization of LAMP2 and Mtb phagosomes increased. Therefore, it was confirmed that OMS-A and OMS-B induce maturation of Mtb phagosomes in macrophages.

8. Within macrophages Mtb Pagosome Necessary for maturation and antibacterial response AMPK on the path of stigma samicin effect

Since it is known that activation of AMP-activated protein kinase (AMPK) induces Mtb phagosome maturation and antibacterial response in macrophages, OMS-A and OMS-B induce activation of AMPK signaling pathway It was confirmed whether or not

As described in 4., OMS-A and OMS-B were added to Mycobacterium tuberculosis-infected macrophages and cultured for about 18 hours. Cell lysates were obtained from cultured cells, anti-p-AMPK catalytic α-subunit (AMPK-α) Thr172 (T172) antibody (Cell signaling), anti-p-acetyl-CoA carboxylase (acetyl- Immunoblotting was performed using a CoA carboxylase: ACC) antibody (Cell signaling) and an anti-β actin antibody (Cell signaling). Immunoblotting images by OMS-A and OMS-B are shown in FIGS. 11A and 11B , respectively.

11a and 11b, OMS-A and OMS-B induced phosphorylation at Thr172 of AMPKα and Ser78 (S79) of acetyl-CoA carboxylase in a time-dependent manner. Specifically, it was confirmed that when macrophages were treated with OMS-A or OMS-B, the AMPK phosphorylation reaction rapidly increased within about 1 hour, then gradually decreased, and then the phosphorylation reaction was induced around 18 hours.

9. of stigma samicin Mitigating effect of Mycobacterium tuberculosis-induced inflammatory response

As described in 4., 1 µM, 5 µM, or 10 µM of OMS-A and OMS-B were added to Mycobacterium tuberculosis-infected macrophages and cultured for about 24 hours.

Cultured macrophages were obtained, and the amounts of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and IL-12 p40 secreted from the macrophages were measured using an ELISA kit (BD ) was measured using.

Figure 12a shows the amount of cytokines secreted from Mycobacterium tuberculosis-infected macrophages according to the amount of Omyeongsamycin (U: untreated, SC: solvent control, ***: p <0.001, **: p <0.01) . As shown in Figure 12a, when OMS-A or OMS-B was treated with Mycobacterium tuberculosis-infected macrophages, the anti-inflammatory cytokines TNF-α, IL-6, IL-1β and IL-12 The amount of p40 decreased in a concentration-dependent manner.

In addition, a vector (Genetransfer Vector Core) containing a nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB)-luciferase reporter gene was transfected into macrophages, and Mycobacterium tuberculosis was infected. Thereafter, 1 μM, 5 μM, or 10 μM of OMS-A and OMS-B were added and cultured for about 6 hours. Thereafter, cell lysates were obtained from macrophages, and the luciferase activity of the cell lysates was measured. The measured luciferase activity is shown in Figure 12b (U: untreated, SC: solvent control, ***: p<0.001, **: p<0.01). As shown in FIG. 12B , when OMS-A or OMS-B was treated with Mycobacterium tuberculosis-infected macrophages, the activity of luciferase was decreased in a concentration-dependent manner, and the expression of NF-κB, an inflammatory factor, was inhibited.

Therefore, it was confirmed that OMS-A and OMS-B could alleviate the inflammatory response of Mycobacterium tuberculosis-infected macrophages.

Claims (20)

The compound represented by Formula 3 or Formula 4, a solvate, or a pharmaceutically acceptable salt thereof, of the genus Mycobacterium (Mycobacterium sp.) Bacterial infection or inflammation caused by infection with bacteria of the genus Mycobacterium Pharmaceutical composition for prophylaxis or treatment:
[Equation 3]
.
[Equation 4]
. delete delete delete delete delete delete delete delete delete The pharmaceutical composition according to claim 1, wherein the bacterial infection of the genus Mycobacterium is selected from the group consisting of tuberculosis, leprosy, and nontuberculous mycobacteria (NTM) infection. The pharmaceutical composition of claim 11, wherein the tuberculosis is multi drug resistance (MDR) or extensive drug resistance (XDR) tuberculosis. delete The pharmaceutical composition according to claim 1, wherein the bacteria of the genus Mycobacterium are Mycobacterium tuberculosis. The method according to claim 1, wherein the compound represented by Formula 3 or Formula 4, a solvate, or a pharmaceutically acceptable salt thereof promotes autophagy of macrophages, relieves inflammation, or induces a combination thereof. A pharmaceutical composition which is. The pharmaceutical composition according to claim 1, further comprising an antibiotic. 17. The pharmaceutical composition according to claim 16, wherein the antibiotic is an anti-tuberculosis agent. The method of claim 17, wherein the anti-tuberculosis agent isoniazid, rifampicin, ethambutol, SQ-109, pyrazinamide, streptomycin, kanamycin, capreomycin ( capreomycin), ethionamide, prothionamide, enviomycin, para-aminosalicylic acid, cycloserine, amikacin, levofloxacin (levofloxacin), moxifloxacin, gatifloxacin, ofloxacin, terizidone, thionamide, ethionamide, protionamide , clofazimine, linezolid, amoxicillin, clavulanate, thioacetazone, imipenem, cilastatin, clarithromycin (clarithromycin), bedaquiline (bedaquiline), delamanid (delamanid), limipenem (lmipenem), silastine (cilastatin), meropenem (Meropenem), or a pharmaceutical composition that is a combination thereof. A method for preventing or treating bacterial infection of the genus Mycobacterium or inflammation caused by bacterial infection of the genus Mycobacterium, comprising administering the pharmaceutical composition of claim 1 to a subject,
Wherein the subject is a non-human mammal. 20. The method of claim 19, wherein the method further comprises administering an antibiotic to the subject.

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