USE OF RIFAMYCIN-QUINOLIZIDONE COUPLING MOLECULE

Abstract

The present invention provides a use of a rifamycin-quinolizidone coupling molecule, or a stereoisomer, hydrate, deuterium-substituted form, ester, solvate, crystal form, metabolite, pharmaceutically acceptable salt or prodrug thereof in resisting nontuberculous mycobacteria. The rifamycin-quinolizidone coupling molecule has a structure shown in formula (I)

##STR00001##

The rifamycin-quinolizidone coupling molecule, or the stereoisomer, hydrate, deuterium, ester, solvate, crystal form, metabolite, pharmaceutically acceptable salt or prodrug thereof may effectively against nontuberculous mycobacteria, and then may be used for treating infection caused by human nontuberculous mycobacteria.

Claims

1. A method of inhibiting or killing nontuberculous mycobacteria, comprising: contacting a rifamycin-quinolizidone coupling molecule having a formula (I) below or a pharmaceutically acceptable salt thereof with nontuberculous mycobacteria. ##STR00004##

2. The method of claim 1, wherein the nontuberculous mycobacteria comprise Mycobacterium avium, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium intracellulare, Mycobacterium massiliense, Mycodacterium chelonei, Mycobacterium fortuitum, or any combinations thereof.

3. The method of claim 1, wherein the nontuberculous mycobacteria comprise Mycobacterium avium, Mycobacterium abscessus, Mycobacterium kansasii, or any combinations thereof.

4. A method of treating infections of nontuberculous mycobacteria, comprising: administrating a pharmaceutical composition comprising: a pharmaceutical acceptable carrier; and a pharmaceutical effective amount of a rifamycin-quinolizidone coupling molecule having a formula (I) below or a pharmaceutically acceptable salt thereof. ##STR00005##

5. The method of claim 4, wherein the nontuberculous mycobacteria comprise Mycobacterium avium, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium intracellulare, Mycobacterium massiliense, Mycodacterium chelonei, Mycobacterium fortuitum, or any combinations thereof.

6. The method of claim 4, wherein the nontuberculous mycobacteria comprise Mycobacterium avium, Mycobacterium abscessus, Mycobacterium kansasii, or any combinations thereof.

7. A pharmaceutical composition for treating nontuberculous mycobacteria infections, comprising: a pharmaceutical acceptable carrier; and a pharmaceutical effective amount of a rifamycin-quinolizidone coupling molecule having a formula (I) below or a pharmaceutically acceptable salt thereof. ##STR00006##

8. The pharmaceutical composition of claim 7, wherein the nontuberculous mycobacteria comprise Mycobacterium avium, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium intracellulare, Mycobacterium massiliense, Mycodacterium chelonei, Mycobacterium fortuitum, or any combinations thereof.

9. The pharmaceutical composition of claim 7, wherein the nontuberculous mycobacteria comprise Mycobacterium avium, Mycobacterium abscessus, Mycobacterium kansasii, or any combinations thereof.

Description

DESCRIPTION OF THE EMBODIMENTS

[0015] To understand the technical features, purpose and advantageous effects of the present invention more clearly, the technical solution of the present invention is described in detail below, but cannot be understood as the limitation of the implementation scope of the present invention. The experimental methods described in the following embodiments are conventional methods unless otherwise specified; and the reagents and materials are commercially available unless otherwise specified.

Embodiment 1

[0016] This embodiment provides a use of a rifamycin-quinolizidone coupling molecule in resisting NTM, the rifamycin-quinolizidone coupling molecule having a structure shown in formula (I)

##STR00003##

[0017] In this embodiment, an inhibitory test is performed on pathogenic bacteria, i.e. Mycobacterium avium, Mycobacterium abscessus and Mycobacterium kansasii belonging to the nontuberculous mycobacteria using the rifamycin-quinolizidone coupling molecule (formula I) of the present invention and positive controls, and then Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) are obtained. Clarithromycin, moxifloxacin, amikacin, rifampicin, rifabutin, ciprofloxacin and metronidazole are used as positive controls. The test strains are provided by KnowBio company, and the Chinese clinically isolated strains are derived from Shanghai Pulmonary Hospital.

[0018] The inhibitory test of this embodiment is performed by the micro broth dilution method using two different culture mediums: (1) MH micro broth dilution method: using MH culture medium (or culture solution), wherein the recommended concentration of calcium and magnesium ions in MH broth (cation-adjusted) is consistent with that in the Guideline of the Clinical and Laboratory Standards Institute (CLSI; M7-A7); and (2) 7H9 micro broth dilution method: using 7H9 culture medium (or culture solution, provided by Sigma-Aldrich). The reason of using two culture mediums, i.e. MH culture medium and 7H9 culture medium to perform composite screening is that the antimycobacterial compound shows different inhibitory activities in different liquid culture mediums, and embodiments of the present invention optimize the drug sensitivity test performed on NTM by using different broth culture mediums in the micro broth dilution method to make it closer to clinical conditions.

[0019] The method for testing MIC comprises the following steps.

[0020] Nontuberculous mycobacteria of rapidly growing mycobacteria (RGM) were grown on a 7H11 agar plate (provided by Sigma-Aldrich) in an air environment at 35-37 C. for about 3 days (depending on bacterial strains). Nontuberculous mycobacteria of slowly growing mycobacteria (SGM) were grown on a 7H11 agar plate (provided by Sigma-Aldrich) in an air environment at 37 C. for 21-30 days;

[0021] Certain colonies were selected from the agar plate, and then placed in the MH or 7H9 culture solution with 0.05% Tween-80. The selected colonies were cultured in an air environment at 35-37 C. for 3 days (rapidly growing) or 12 days (slowly growing) until the absorbance (OD value) thereof reached 0.08-0.1 (0.5 Mcfarland Standard). A bacterial suspension with the absorbance (OD600 value) of 0.08-0.1 (0.5 Mcfarland Standard) was prepared with normal saline.

[0022] 180 L of broth (MH or 7H9 culture solution) was added into the first column of holes of a 96-well plate. 100 L of broth (MH or 7H9 culture solution) was added into other holes of the 96-well plate. The compound of formula I was formulated into 1.28 mg/mL of solution using DMSO to be tested in the range of 64-0.062 g/mL immediately. 20 L of compound was added into the first column of holes, and 100 L thereof was taken for continuous dilution. Finally, 100 L of nontuberculous mycobacteria strain suspension was added into all holes except the culture medium control holes, wherein different quality control reagents are added for different microorganisms. These holes include: 1) negative control holes only containing bacteria; 2) negative control holes only containing culture medium; 3) positive control holes containing clarithromycin and the like; and 4) control holes containing optional Escherichia coli.

[0023] For RGM, the OD value was assayed on the third day, and for SGM, the OD value was assayed on the twelfth day. Assay was performed by using the method of resazurin microtiter assay plate recommended by the Clinical and Laboratory Standards Institute. In short, the method was to add resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) into the 96-well plate. Resazurin is a blue dye, has weak fluorescence, may be irreversibly reduced to pink or highly red fluorescent dye; and may be used as a redox indicator when testing the MIC of living bacteria.

[0024] The method for testing MBC comprises the following steps.

[0025] A culture solution having a concentration equal to the MIC and a culture solution having a concentration higher than the MIC hole concentration (dilution of 0-1-2-3-4-5-6-7) were coated on 7H11 or MH agar plates in quadruplicate (four plates/holes), and then cultured in an air environment at 35-37 C. (depending on bacterial strains) to calculate CFU. wherein MIC.sub.90 is the minimum drug concentration for inhibiting 90% of NTM isolated strains, and MBC.sub.99 is the minimum drug concentration for killing 99.99% of initial bacteria.

[0026] The test results are shown in Tables 1-3:

TABLE-US-00001 TABLE 1 Minimum Inhibitory Concentration (MIC, mcg/mL) of Rifamycin-Quinolizidone Coupling Molecule (Formula I) Against Nontuberculous Mycobacteria 7H9 Broth MH Broth Mycobacterium Mycobacterium avium Mycobacterium Mycobacterium avium Mycobacterium Mycobacterium Compound Smooth Rough abscessus kansasii Smooth Rough abscessus kansasii Compound I 4 >64 1 2 4 >64 1 2 Clarithromycin 32 >64 16 2 >64 >64 64 2 Amikacin 2 >64 32 16 2 2 16 1 Rifampicin 0.5 8 >64 16 1 8 >64 16 Ciprofloxacin >64 ND 16 >64 >64 ND >64 >64 Metronidazole >64 ND >64 >64 >64 ND >64 >64

TABLE-US-00002 TABLE 2 Minimum Bactericidal Concentration (MBC, mcg/mL) of Rifamycin-Quinolizidone Coupling Molecule (Formula I) Against Nontuberculous Mycobacteria 7H9 Culture Medium MH Culture Medium Mycobacterium Mycobacterium avium Mycobacterium Mycobacterium avium Mycobacterium Mycobacterium Compound Smooth Rough abscessus kansasii Smooth Rough abscessus kansasii Compound I 16 >64 1 2 16 >64 1 2 Clarithromycin >64 >64 >64 64 >64 >64 >64 >64 Amikacin >64 >64 >64 64 >64 >64 >64 >64 Rifampicin 4 >64 >64 4 4 ND >64 4 Ciprofloxacin >64 ND >64 >64 >64 ND >64 >64 Metronidazole >64 ND >64 >64 >64 ND >64 >64

TABLE-US-00003 TABLE 3 Minimum Inhibitory Concentration (MIC, mcg/mL, MH Culture Medium) of Rifamycin-Quinolizidone Coupling Molecule (Formula I) Against Isolated Strains from Chinese Hospital Clinically Isolated Strains Rifampicin Moxifloxacin Clarithromycin Rifabutin Metronidazole Compound I Mycobacterium Avium (standard 0.25 1 0.0625 0.0625 8 1 avium strain) Avium (S31) 0.0625 8 0.125 0.0625 16 0.25 Avium (S123) 8 0.5 0.5 0.0625 8 8 Avium (S559) 0.125 1 0.25 0.0625 8 0.25 Avium (S568) 4 0.5 4 0.0625 8 32 Avium (S577) 0.125 0.5 0.25 0.0625 8 0.125 Avium (S597) 8 2 1 0.0625 8 >64 Avium (S602) 2 4 1 0.0625 32 8 Mycobacterium Kansasii 0.125 0.0625 0.0625 0.0625 8 0.0625 kansasii (standard strain) Kansasii (S9) 0.125 0.0625 0.125 0.0625 8 0.25 Kansasii (S199) 0.0625 0.0625 0.0625 0.0625 8 0.0625 Kansasii (S520) 0.0625 0.0625 0.0625 0.0625 8 0.0625 Kansasii (S609) 0.0625 0.0625 0.125 0.0625 8 0.0625 Mycobacterium Intracellulare 0.25 0.25 0.0625 0.0625 8 0.5 intracellulare (standard strain) Intracellulare (S90) 8 2 1 0.125 16 64 Intracellulare (S261) 16 2 1 0.25 16 64 Intracellulare (S269) 4 2 1 0.0625 16 >64 Intracellulare (S290) 4 2 1 0.0625 8 64 Intracellulare (S291) 4 1 1 0.0625 8 64 Intracellulare (S298) 4 2 2 0.5 16 64 intracellulare (S442) 2 2 2 0.0625 8 32 Intracellulare (S541) 16 64 64 0.125 16 32 Intracellulare (S623) 8 2 4 0.25 8 64 Intracellulare (ZPS) 2 1 1 0.0625 8 32 Mycobacterium Massiliense (D10) >64 32 0.25 >64 16 >64 massiliense Massiliense (D14) >64 32 0.0625 64 16 >64 Massiliense (D18) >64 8 0.0625 64 8 >64 Mycobacterium Fortuitum >64 1 32 4 16 64 fortuitum (standard strain) Fortuitum (S103) >64 1 >64 32 8 >64 Fortuitum (S111) 64 0.5 2 4 16 >64 Mycodacterium Chelonei >64 4 0.5 64 16 64 chelonei (standard strain)

[0027] As shown in the above Tables 1, 2 and 3, the minimum inhibitory concentrations (MIC) of the rifamycin-quinolizidone coupling molecule (formula I) of the present invention against smooth type strain and rough type strain of Mycobacterium avium were similar to that of amikacin, and were superior to that of clarithromycin. The activity of the compound I against mycobacterium kansasii was much higher than that of rifampicin, ciprofloxacin and metronidazole, and was similar to that of other control compounds; and the activity of the compound against Mycobacterium intracellulare was weak. For the minimum bactericidal concentration (MBC), the bactericidal activity of the rifamycin-quinolizidone coupling molecule (formula I) against Mycobacterium kansasii was higher than or much higher than that of all other tested antibiotic drugs; and the rifamycin-quinolizidone coupling molecule also had an activity against smooth type strain of Mycobacterium avium. Meanwhile, the two different culture mediums had substantially identical test results, and can be suitable for clinical application. The results of research show that the rifamycin-quinolizidone coupling molecule of the present invention (formula I) had an effective and broad in-vitro activity against NTM, and thus may be used for treating human NTM infections.

[0028] In addition, the stereoisomer, hydrate, deuterium-substituted form, ester, solvate, crystal form, metabolite, pharmaceutically acceptable salt or prodrug of the rifamycin-quinolizidone coupling molecule of embodiments of the present invention may be used for resisting nontuberculous mycobacteria as well, and may be used for preparing a drug for treating a disease induced by human nontuberculous mycobacteria infections.

[0029] In another specific embodiment, the stereoisomer, hydrate, deuterium-substituted form, ester, solvate, crystal form, metabolite, pharmaceutically acceptable salt or prodrug of the rifamycin-quinolizidone coupling molecule may be combined with conventional antibacterial drugs in the art as well, to be used for treating a disease caused by NTM infections.