PHARMACEUTICAL COMPOSITION CONTAINING MACROLIDE COMPOUND, PRODUCTION METHOD THEREFOR, AND METHOD USING SAME

Abstract

A pharmaceutical composition or a health functional food including a macrolide compound, a method of preparing the same, and a method using the same may be used to prevent or treat Mycobacterium sp. bacterial infection, such as tuberculosis, Hansen's disease, and non-tuberculous Mycobacterium sp. bacterial infections, or symptoms related thereto.

Claims

1. A pharmaceutical composition for preventing or treating Mycobacterium sp. bacterial infection or symptoms related thereto, comprising a compound or a stereoisomer, solvate, or pharmaceutically acceptable salt thereof, wherein the compound is represented by Formula 1: ##STR00006## wherein in Formula 1, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.7, R.sup.8, and R.sup.9 are each independently selected from hydrogen, a hydroxyl group, an amino group, a halogen group, a ketone group, a cyano group, a substituted or unsubstituted C.sub.1 to C.sub.20 alkyl group, a substituted or unsubstituted C.sub.1 to C.sub.20 alkoxy group, a substituted or unsubstituted C.sub.2 to C.sub.20 alkenyl group, a substituted or unsubstituted C.sub.2 to C.sub.20 alkynyl group, or a combination thereof, R.sup.5 and R.sup.6 are each independently hydrogen, a hydroxyl group, an amino group, a halogen group, a cyano group, —C(═O)R.sub.a, —C(═O)OR.sub.a, —OCO(OR.sub.a), —C═N(R.sub.a), —SR.sub.a, —S(═O)R.sub.a, —S(═O).sub.2R.sub.a, —PR.sub.a, a substituted or unsubstituted C.sub.1 to C.sub.20 alkyl group, a substituted or unsubstituted C.sub.1 to C.sub.20 alkoxy group, a substituted or unsubstituted C.sub.2 to C.sub.20 alkenyl group, a substituted or unsubstituted C.sub.2 to C.sub.20 alkynyl group, a C.sub.2 to C.sub.20 alkylene oxide group, a substituted or unsubstituted C.sub.3 to C.sub.30 cycloalkyl group, a substituted or unsubstituted C.sub.6 to C.sub.30 aryl group, a substituted or unsubstituted C.sub.6 to C.sub.30 aryloxy group, a substituted or unsubstituted C.sub.6 to C.sub.30 heteroaryl group, or a combination thereof, wherein R.sub.a is hydrogen, a C.sub.1 to C.sub.10 alkyl group, or a C.sub.6 to C.sub.20 aryl group, n is an integer from 1 to 20, X is a substituted or unsubstituted 3-membered to 10-membered heterocyclic ring group containing one or more O, and the substituted heterocyclic ring group is substituted with a hydroxyl group, a C.sub.1 to C.sub.10 alkyl group, a C.sub.1 to C.sub.10 alkoxy group, or a combination thereof, and Y is a substituted or unsubstituted C.sub.1 to C.sub.30 alkyl group, and the substituted alkyl group is substituted with one or more hydroxyl groups or C.sub.1 to C.sub.10 alkoxy groups.

2. The pharmaceutical composition of claim 1, wherein at least one of R.sup.1, R.sup.3, and R.sup.8 is a substituted or unsubstituted C.sub.1 to C.sub.10 alkoxy group.

3. The pharmaceutical composition of claim 2, wherein at least one of R.sup.1, R.sup.3, and R.sup.8 is a methoxy group.

4. The pharmaceutical composition of claim 1, wherein at least one of R.sup.2, R.sup.4, R.sup.7, and R.sup.9 is a hydroxyl group.

5. The pharmaceutical composition of claim 1, wherein at least one of R.sup.5 and R.sup.6 is a methyl group.

6. The pharmaceutical composition of claim 1, wherein the heterocyclic ring group in X is an ethylene oxide group or tetrahydrofuran.

7. The pharmaceutical composition of claim 1, wherein the compound represented by Formula 1 is represented by Formula 2 or Formula 3: ##STR00007##

8. The pharmaceutical composition of claim 1, wherein the compound represented by Formula 1 is represented by Formula 4 or Formula 5: ##STR00008##

9. The pharmaceutical composition of claim 1, wherein the Mycobacterium sp. bacterial infection is selected from the group consisting of tuberculosis, Hansen's disease, and nontuberculous mycobacterial (NTM) infection.

10. The pharmaceutical composition of claim 1, wherein the bacteria of the Mycobacterium sp. is selected from the group consisting of Mycobacterium tuberculosis, nontuberculous mycobacteria, and a BCG strain.

11. The pharmaceutical composition of claim 1, wherein the bacteria of the Mycobacterium sp. is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium microti, Mycobacterium leprae, Mycobacterium canetti, Mycobacterium avium, Mycobacterium kansasii, and Mycobacterium marinum.

12. The pharmaceutical composition of claim 1, further comprising an antibiotic.

13. The pharmaceutical composition of claim 12, wherein the antibiotic is an anti-tuberculosis drug.

14. The pharmaceutical composition of claim 13, wherein the anti-tuberculosis drug is ethambutol, isoniazid, rifampicin, SQ-109, pyrazinamide, streptomycin, kanamycin, capreomycin, ethionamide, prothionamide, enviomycin, para-aminosalicylic acid, cycloserine, amikacin, levofloxacin, moxifloxacin, gatifloxacin, ofloxacin, terizidone, thionamide, ethionamide, prothionamide, clofazimine, linezolid, amoxicillin, clavulanate, thioacetazone, imipenem, cilastatin, clarithromycin, bedaquiline, delamanid, limipenem, cilastatin, meropenem, or a combination thereof.

15. A Micromonospora sp. GR10 strain (Accession No.: KCTC14124BP) for producing the compound represented by Formula 1, or the stereoisomer, solvate, or pharmaceutically acceptable salt thereof, of claim 1.

16. A method of manufacturing the compound represented by Formula 1, or the stereoisomer, solvate, or pharmaceutically acceptable salt thereof, of claim 1, or the pharmaceutical composition comprising the same, the method comprising: culturing a Micromonospora sp. GR10 strain (accession number: KCTC14124BP); and isolating the compound represented by Formula 1, or the stereoisomer, solvate, or pharmaceutically acceptable salt thereof, of claim 1 from a culture of the strain.

17. A health functional food for preventing or ameliorating Mycobacterium sp. bacterial infection or symptoms related thereto, comprising the compound represented by Formula 1 or a stereoisomer, solvate, or salt thereof, of claim 1.

18. A method of preventing or treating Mycobacterium sp. bacterial infection or symptoms related thereto, comprising administering to a subject the pharmaceutical composition of claim 1.

19. The method of claim 18, further comprising administering an antibiotic to the subject.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0078] FIG. 1 is a photograph of a culture medium of Micromonospora sp. GR10 strain.

[0079] FIG. 2A is a schematic of a method for confirming the Mycobacterium tuberculosis-removing activity of arenicolide A; FIG. 2B is a graph showing the number of viable cells of Mycobacterium tuberculosis in lung tissue when arenicolide A is administered (CFU: colony forming unit); and FIG. 2C is a dose response curve of arenicolide A for a tuberculosis strain (RFU: relative fluorescence unit).

[0080] FIG. 3 is a graph showing the number of viable cells of a BCG strain in lung tissue when arenicolide A or arenicolide C is administered (CFU: colony forming unit).

[0081] FIG. 4 is a graph showing the number of viable cells of an M. abscessus strain in lung tissue when arenicolide A is administered (CFU: colony forming unit).

[0082] FIG. 5A is a fluorescence image showing the inhibition of growth of Mycobacterium tuberculosis according to concentrations of arenicolide A and ethambutol; and FIG. 5B is an image of Mycobacterium tuberculosis cultured in a solid medium containing arenicolide A and ethambutol.

MODE OF DISCLOSURE

[0083] Hereinafter, the disclosure will be described in more detail through embodiments. However, these embodiments are intended to illustrate the present disclosure, and the scope of the present disclosure is not limited to these embodiments.

Example 1. Isolation of Arenicolide and Structural Identification

[0084] 1-1. Isolation of Arenicolide-Producing Strains

[0085] The arenicolide producing strain of GR10 strain was isolated from K solid medium (25 g of starch, 2 g of yeast extract, 15 g of soytone, 4 g of CaCO.sub.3, and 20 g of agar per 1 L of distilled water) (FIG. 1).

[0086] The whole genome of GR10 strain was isolated and 16S ribosomal DNA was cloned by polymerase chain reaction (PCR), and the nucleic acid sequence was analyzed (SEQ ID NO: 1). Based on the results of 16S rDNA sequencing, the strain was identified as the Micromonospora genus. This strain was named Micromonospora sp. GR10 strain, and the strain was deposited to the Korea Research Institute of Bioscience and Biotechnology on Jan. 31, 2020 (Accession No.: KCTC14124BP).

[0087] 1-2. Culture of GR10 Strain of Micromonospora Genus

[0088] The GR10 strain of the Micromonospora genus was spread on a sterile YEME solid medium (4 g of yeast extract, 10 g of malt extract, 4 g of glucose, and 20 g of agar per 1 L of distilled water) and cultured for 4 weeks at approximately 30° C.

[0089] The spores of the cultured GR10 strain were inoculated into 150 mL of modified K liquid medium (2 g yeast extract, 5 g malt extract, 5 g soytone, 5 g starch, 2 g starch, 5 g of mannitol, and 6 g of glycerol per 1 L distilled water), and cultured for about 7 days at 30° C., with shaking at 200 rpm. 5 mL of the culture medium was inoculated into a 200 mL volume of modified K liquid medium, and cultured for about 4 days under the conditions of 170 rpm and 30° C. Then, 10 mL of the culture medium was inoculated into a 1 L volume of modified K liquid medium, and cultured for about 7 days under the conditions of 170 rpm and 30° C.

[0090] 1-3. Isolation and Purification of Arenicolide

[0091] The culture of the GR10 strain cultured in Example 1-2 was obtained.

[0092] 1 L of the culture medium of the cultured GR10 strain and about 1 L volume of ethyl acetate (EtOAc, Daejeong Hwageum Co., Ltd.) were put into a separatory funnel mounted on a stand, then the funnel was closed with a stopper and shaken up, down, left and right for 1 minute to proceed the first extraction. After that, the funnel was mounted on a stand again, and after the water layer and the EtOAc layer were completely separated, the valve of the separatory funnel was opened to remove the water layer. Fresh EtOAc was added to the aqueous layer, and repeated extraction was performed in the same manner. The EtOAc layer was separately stored in a clean flask, and anhydrous sodium sulfate (Daejeong Hwageum Co., Ltd.) was added to remove the water remaining in the culture. The EtOAc layer from which the water was removed was transferred back to a 3 L round flask, and then dried under reduced pressure using a decompression dryer. A total of 50 L of the strain was cultured, and as a result of the extraction, about 10 g of crude extract was obtained.

[0093] High-performance liquid chromatography (HPLC) was used to purify arenicolide from the GR10 strain extract. The extract was dissolved in a sufficient amount of methanol (MeOH), and solid impurities were removed by using a syringe filter (ADVANTEC, 25HP045AN), and a purification process was proceeded by using preparative HPLC. For material purification, a reversed-phase column (C.sub.18(2) Luna 10 μm 250×21.2 mm) was used, and a condition of a concentration gradient (flow rate: 10 mL/min, detection: UV 230 nm) of acetonitrile (ACN)/aqueous solution of 30% (v/v) to 70% (v/v) was used.

[0094] It was confirmed through liquid chromatography mass spectrometry (LC/MS) analysis that pure arenicolide A was eluted about 32 minutes after injection into HPLC under these conditions. Through repeated experiments, 250 mg of arenicolide A and 50 mg of arenicolide C were obtained from 10 g of extract.

[0095] 1-4. Analysis of Physicochemical Properties of Arenicolide

[0096] (1) Identification of Chemical Structure of Arenicolide A

[0097] The structure of arenicolide A was identified based on 1 D and 2D nuclear magnetic resonance (NMR) spectra. For nuclear magnetic resonance spectra (.sup.1H NMR, .sup.13C NMR), 500 MHz NMR manufactured by Bruker was used, and DMSO-d.sub.6 was used as the solvent.

[0098] The structural positioning of arenicolide A by nuclear magnetic resonance spectra is shown in Table 1.

[0099] [Arenicolide A]

[0100] (1) Molecular formula: C.sub.45H.sub.72O.sub.12

[0101] (2) Molecular weight: 804

[0102] (3) Color: Transparent

[0103] (4).sup.1H-NMR (DMSO-d.sub.6, 600 MHz): see Table 1

[0104] (5).sup.13C-NMR (DMSO-d.sub.6, 150 MHz): see Table 1

TABLE-US-00001 TABLE 1 Position δ.sub.C Type δ.sub.H mult. (J in Hz) 1 166.6 C 2 121.6 CH 5.95 d (15.0) 3 143.9 CH 7.23 dd (15.0, 11.0) 4 132.6 CH 6.39 dd (15.0, 11.0) 5 139.4 CH 5.84 dd (15.0, 8.0) 6 85.7 CH 3.58 dd (8.0, 8.0) 7 75.6 CH 3.96 dd (8.0, 8.0) 8 124.4 CH 5.32 dd (15.0, 8.0) 9 139.3 CH 6.15 d (15.0) 10 131.5 C 11 140.2 CH 5.23 d (9.0) 12 32.3 CH 2.50 m 13 37.0 CH.sub.2 1.24 m 14 23.1 CH.sub.2 1.30 m 15 29.9 CH.sub.2 1.45 m 16 84.8 CH 3.05 m 17 74.6 CH 4.00 dd (8.0, 5.0) 18 127.4 CH 5.60 dd (15.0, 8.0) 19 136.6 CH 6.18 d (15.0) 20 133.0 C 21 135.2 CH 5.24 d (9.0) 22 36.6 CH 2.77 m 23 76.0 CH 3.29 m 24 82.7 CH 3.30 m 25 77.9 CH 5.39 m 26 127.6 CH 5.82 m 27 130.7 CH 5.81 m 28 36.2 1.30 2.34/2.16 m 29 76.1 CH 3.21 dd (9.0, 3.0) 30 61.9 C 31 62.2 CH 2.88 d (9.0) 32 83.1 CH 2.99 dd (8.5, 5.5) 33 71.0 CH 3.55 m 34 34.8 CH.sub.2 1.53/1.42 m 35 18.7 CH.sub.2 1.54/1.34 m 36 13.1 CH.sub.3 0.93 t (7.0) 37 55.9 CH.sub.3 3.30 s 38 11.6 CH.sub.3 1.68 s 39 19.7 CH.sub.3 0.94 d (6.5) 40 57.8 CH.sub.3 3.37 s 41 11.7 CH.sub.3 1.77 s 42 16.6 CH.sub.3 1.01 d (7.0) 43 59.4 CH.sub.3 3.38 s 44 12.2 CH.sub.3 1.29 s 45 57.5 CH.sub.3 3.44 s

[0105] (2) Identification of Three-Dimensional Structure of Arenicolide A

[0106] Most of the three-dimensional structure of arenicolide A was revealed in the paper in which arenicolide was first reported (Williams et al., J. Org. Chem. 2007, vol. 72, No. 14, pp. 5025-5034). In order to identify the three-dimensional structure of carbon 39, which has not yet been revealed, a new computational method, DP4 analysis, was used.

[0107] First, since the DP4 analysis method has a higher significance as the molecular weight is lowered, the chain portion, which is judged not to significantly affect the chemical shift of the substance, is excluded, and only the macrocyclic structure part was modeled through energy minimization by using the Avogadro 1.2.0. program, and molecular models having structures 39R and 39S were modeled. And, from among the many chemical forms searched through the stable chemical forms of each of the two models by using the Maestro program, only forms with a stable energy of 10 kJ/mol were selected. The weight of each chemical form was calculated by calculating Boltzmann population according to the relative energy of each of the selected forms. As a result, 8 chemical forms of the 39R model were determined and 36 forms of 39S were determined. Optimization of ground state geometrical structure was performed using each chemical model with the Turbomole X 4.3.2 program, and then, the chemical shift values in each chemical form of the obtained optimized structural models was deduced by calculation. By assigning weight to the models according to the Boltzmann population, the average chemical shift values were calculated. When comparing the chemical shift values revealed by actual experiments with the calculated chemical shift values of 39R and 39S models, the carbon chemical shift value was 96.7%, the hydrogen chemical shift value was 100.0%, and when the carbon and hydrogen values were combined, the chemical shift value was 100.0%, thus the three-dimensional structure of carbon 39 was confirmed to be S.

[0108] The chemical structural formula of arenicolide A analyzed from the nuclear magnetic resonance spectrum data and results of the three-dimensional structural analysis is shown below:

##STR00004##

[0109] On the other hand, the chemical structural formula of arenicolide C is shown below:

##STR00005##

Example 2. Confirmation of Anti-Mycobacterial Activity of Arenicolide

[0110] 2-1. Confirmation of Anti-Tuberculosis Activity of Arenicolide

[0111] C57BL/6 mice (n=16) were prepared to investigate the anti-tuberculous activity of arenicolide A. Mice were nasally infected with 2×10.sup.4/CFU of Mycobacterium tuberculosis (Mtb), and the mice were randomly divided into two groups (each, n=8). From the 4th day, arenicolide A was orally administered at a dose of 100 mg/body weight kg for 6 days. Phosphate-buffered saline (PBS) was administered as a negative control. Mice were sacrificed on day 10 from the day of infection (FIG. 2A). The number of viable Mycobacterium tuberculosis in the lung tissue was investigated, and the results are shown in FIG. 2B (***: p<0.001).

[0112] As shown in FIG. 2B, the number of viable Mycobacterium tuberculosis in the lung tissue was significantly reduced in the group to which arenicolide A was orally administered compared to the negative control group. Therefore, it was confirmed that arenicolide A showed efficacy as a therapeutic candidate in a mouse tuberculosis infection model.

[0113] To know the minimal inhibitory concentration (MIC) of arenicolide A, a dose-response curve (DRC) was calculated using the M. tuberculosis mc2 6230 strain. MIC was measured by using a resazurin microtiter assay (REMA). Briefly, 100 μl of 7H9 liquid medium was added to each well of a 96-well microtiter plate, and two-fold serial dilutions of antibiotics were directly added to each well. Plates were covered and incubated for 5 days at 37° C. Resazurin (0.01% (w/v)) was added to each well and the plates were incubated overnight. Fluorescence intensity was measured by using a SpectraMax M3 Multi-Mode Microplate Reader (Molecular Devices, CA, USA) (ex. 560/em. 590 nm). MIC values were calculated by using Prism 6 (GraphPad Software, Inc., La Jolla, Calif.), and the results are shown in FIG. 2C. As shown in FIG. 2C, the MIC.sub.50 value of arenicolide A was about 24.6 μM.

[0114] On the other hand, as a result of measuring the anti-tuberculosis activity against Mycobacterium tuberculosis Mtb H37Rv strain (standard strain) in the same way, the MIC.sub.50 value of arenicolide A was about 6.9 μM.

[0115] Therefore, arenicolide A was confirmed to have anti-tuberculosis activity.

[0116] 2-2. Confirmation of Anti-BCG Activity of Arenicolide

[0117] It was confirmed whether the arenicolide compound has an effective inhibitory activity against BCG (bacillus Calmette-Guerin) strain, that is, Mycobacterium bovis.

[0118] As described in Example 2-1, 57BL/6 mice were nasally infected with 1×10.sup.6/CFU of BCG strain. From the 4th day, arenicolide A or arenicolide C was orally administered at a dose of 100 mg/body weight kg for 6 days. As a negative control, 2.5% (v/v) DMSO and 2.5% (v/v) ethanol were administered. Mice were sacrificed on day 10 from the day of infection. The number of viable tubercle bacilli in the lung tissue was investigated, and the results are shown in FIG. 3 (each, n=6, **: p<0.01, ***: p<0.001).

[0119] As shown in FIG. 3, it was confirmed that arenicolide A and arenicolide C had effective inhibitory activity against BCG bacteria compared to the negative control.

[0120] 2-3. Confirmation of Anti-Atypical Tubercle Bacilli Activity of Arenicolide

[0121] It was confirmed whether the arenicolide compound has an effective inhibitory activity against Mycobacterium abscessus, atypical tubercle bacilli.

[0122] As described in Example 2-2, 57BL/6 mice were nasally infected with 1×10.sup.7/CFU of M. abscessus strain. From the 3rd day, arenicolide A was orally administered at a dose of 100 mg/body weight kg for 5 days. As a negative control, 2.5% (v/v) DMSO and 2.5% (v/v) ethanol were administered. Mice were sacrificed on day 7 from the day of infection. The number of viable tubercle bacilli in the lung tissue was investigated, and the results are shown in FIG. 3 (each, n=6, ***: p<0.001).

[0123] As shown in FIG. 4, the number of viable cells in the lung tissue was significantly decreased in the group to which arenicolide A was orally administered compared to the negative control group. Arenicolide A was confirmed to be effective as a therapeutic candidate in a mouse non-tuberculous Mycobacterium infection model.

[0124] 2-4. Evaluation of Effectiveness of Combined Administration of Arenicolide and Anti-Tuberculosis Drugs

[0125] The effect by the combined administration was confirmed when arenicolide and an anti-tuberculosis drug were combined.

[0126] Specifically, M. tuberculosis was cultured at 37° C. in Middlebrook 7H9 medium to which ADC was added. A 100 μl of medium aliquot was added to each well of a 96-well microtiter plate, and two-fold serial dilutions of arenicolide A or ethambutol were added directly to each well. The plates were then incubated at 37° C. for 5 days. Resazurin (0.025% (w/v)) was added to each well, and fluorescence intensity was measured by using a SpectraMax® M3 Multi-Mode Microplate Reader (Molecular Devices, CA, USA) (ex/em 560/590 nm). MIC values were calculated by using Prism 6 (GraphPad Software, Inc., La Jolla, Calif.). Fluorescence intensities of different concentrations of arenicolide and ethambutol are shown in FIG. 5A.

[0127] Meanwhile, Mycobacterium tuberculosis was cultured in a solid medium containing arenicolide and ethambutol combined at each MIC. In order to read the survival of the bacteria, Mycobacterium tuberculosis was inoculated into a solid medium (7H10 added), and the growth of the bacteria was measured after culturing at 37° C. for 3 weeks. An image of the cultured bacteria is shown in FIG. 5B.

[0128] As shown in FIGS. 5A and 5B, it was confirmed that the combination of arenicolide A and ethambutol acts synergistically against Mycobacterium tuberculosis. In particular, it was confirmed that colonies of Mycobacterium tuberculosis were not formed in the combination of 1.6 μM of ethambutol and 0.048 μM of arenicolide A and in the combination of 0.8 μM of ethambutol and 0.19 μM of arenicolide A. Through this, it was confirmed that the MIC value of ethambutol, which was administered in combination, may be lowered to about ¼ when a small amount of arenicolide A was administered. In addition, it was confirmed that, when administered alone, arenicolide A (MIC=0.78 μM) has superior efficacy compared to ethambutol (MIC=3.2 μM), a positive control drug.

[0129] <Accession number>

[0130] Name of the deposited institution: Korea Research Institute of Bioscience and Biotechnology

[0131] Accession number: KCTC14124BP

[0132] Deposition date: 20200131