Bactericidal Activities of the Pyrrole Derivative BM212 against Multidrug-Resistant and Intramacrophagic Mycobacterium tuberculosis Strains

Abstract

The pyrrole derivative BM212 [1,5-diaryl-2-methyl-3-(4-methylpiperazin-1-yl)methyl-pyrrole] was shown to possess strong inhibitory activity against both Mycobacterium tuberculosis and some nontuberculosis mycobacteria. BM212 was inhibitory to drug-resistant mycobacteria and also exerted bactericidal activity against intracellular bacilli residing in the U937 human histiocytic lymphoma cell line.

The frequent appearance of multidrug-resistant strains of Mycobacterium tuberculosis and the growing importance of nontuberculosis mycobacterial (NTM) strains in infections of immunosuppressed patients have accentuated the need to search for new antimycobacterial drugs (6, 7, 10, 12, 14, 18, 19). Recently, among the various active compounds already discovered, some azole derivatives have been shown to possess strong inhibitory activities in vitro and in vivo against M. tuberculosis strains (2). In addition, metronidazole was found to be able to kill dormant cells of M. tuberculosis (22).

With the aim of finding new more potent antimycobacterial drugs, we tested several azole compounds containing the imidazole, pyrrole, toluidine, or methanamine group (3, 8, 9). Among these compounds the pyrrole derivative BM212 appeared to be endowed with particularly potent and selective antimycobacterial properties, and consequently, we devised some experiments in order to characterize its activity against both drug-resistant and intramacrophagic mycobacteria. BM212 is a 1,5-diaryl-2-methyl-3-(4-methylpiperazin-1-yl)methyl-pyrrole, and its formula is indicated in Fig. 1 (5). Isoniazid (INH) and streptomycin (SM) were used as controls.

FIG. 1.

Chemical structure of BM212 [1,5-diaryl-2-methyl-3-(4-methylpiperazin-1-yl)-methyl-pyrrole].

Mycobacterial strains and MIC determinations.

A total of seven mycobacterial strains were purchased from the Institut Pasteur Collection (CIP, Paris, France) (Table 1). The other strains used were of clinical origin and were identified by conventional methods (17). The MICs of BM212 and the controls were determined for several strains of M. tuberculosis and nontuberculous mycobacteria by the BACTEC 460 TB method (11, 13). A broth microdilution assay was used for rapidly growing strains (4). Several drug-resistant M. tuberculosis strains of clinical origin were isolated from the University hospitals of Cagliari and Sassari, Italy. Their drug resistance was detected by standard procedures (13). The MICs of BM212 for 14 clinical isolates of M. tuberculosis, which tested resistant to some of the most commonly used antimycobacterial drugs, were determined by the BACTEC 460 TB technique according to the method of Lee and Heifets (15).

TABLE 1.

Inhibitory activities of BM212 against various species of mycobacteria

Strain (no. of clinical strains)	MIC (μg/ml)
	BM212	INH	SM
M. tuberculosis CIP6431	1.5	0.1	1.5
M. tuberculosis CIP103471	0.7	0.2	0.7
M. tuberculosis CIP6425	1.5	0.4	1.5
M. tuberculosis (19)	0.7–6.2	0.05–0.2	0.4–6.2
M. fortuitum CA10	3.1	12.5	25
M. fortuitum (8)	3.1–12.5	12.5–50	25–50
M. smegmatis CIP103599	25	50	6.2
M. smegmatis (6)	3.1–25	50–>100	6.2–25
M. marinum CIP6423	100	12.5	25
M. gordonae CIP6427	>100	25	12.5
M. gordonae (6)	6.2–>100	25–50	12.5–50
M. avium CIP103317	0.4	25	6.2
M. avium (14)	0.4–3.1	25–>100	6.2–25
M. kansasii (4)	3.1–6.2	6.2–25	12.5–50

The pyrrole derivative BM212 showed potent antimycobacterial activities against several strains of M. tuberculosis (Table 1). The MICs were between 0.7 and 1.5 μg/ml for both collection and clinical strains; for only one strain was the MIC as high as 6.2 μg/ml. These values were a little higher than those of INH (0.05 to 0.2) for most strains but were generally comparable with those of SM (from 0.4 to 6.2 μg/ml). Also, some NTM strains appeared to be quite susceptible to the action of BM212. In fact, the MIC ranges were 3.1 to 12.5 μg/ml for M. fortuitum, 3.1 to 25 μg/ml for M. smegmatis, and 3.1 to 6.2 for M. kansasii, while for M. avium, it was between 0.4 and 3.1 μg/ml. M. marinum (a single strain) and M. gordonae appeared less susceptible to the inhibiting activity of BM212.

The activity of BM212 against various drug-resistant mycobacteria was tested. Two strains were only resistant to ethambutol (EMB), three were resistant to amikacin (AMK), two were resistant to SM, two were resistant to INH, and two were resistant to both rifampin (RIF) and rifabutin (RIB). Two strains were resistant to both INH and RIF, and strain MSS3 was highly resistant to four drugs (INH, EMB, RIF, and RIB). BM212 had inhibitory activity against all strains tested, with MICs between 0.7 and 1.5 μg/ml. The BM212 MIC for one strain of AMK-resistant mycobacterium was as high as 6.2 μg/ml.

Bactericidal activity of BM212 against intracellular mycobacteria.

The bactericidal activity of BM212 against intracellular mycobacteria was studied using U937 cells (ICN-FLOW), a human histiocytic cell line (21), grown in RPMI 1640 medium with 10% fetal calf serum (1, 16, 20). In six multiwell plates, 2 × 10⁶ cells for each well were seeded in the presence of 20 ng of phorbol myristate acetate/ml. The cells were incubated at 36°C with 5% CO₂. Within 72 h, the U937 cells adhered to the well bottom and differentiated into macrophages. A suspension of M. tuberculosis CIP103471 containing 10⁶ bacilli/ml of RPMI 1640 was prepared from an actively growing culture. Two milliliters of this suspension was left for 4 h on the cell monolayer, and then the culture was washed four times, in order to remove extracellular bacilli. At the end of infection some plates were processed for counting the number of bacilli internalized by the cells. BM212 in concentrations ranging from 10 to 0.5 μg/ml was added to the cultures (in triplicate), which were then incubated for 7 days in an atmosphere of 5% of CO₂. At the end of the incubation period, the cells were washed again with fresh Hanks’ balanced salt solution, detached from the plates, and counted; subsequently they were lysed with Dulbecco’s modified phosphate buffer (ICN-FLOW) containing 0.25% sodium dodecyl sulfate. The lysed cells were sonicated for 20 s, and the bacilli were titered on 7H11 agar plates with 10% OADC (Difco).

The tubercle bacilli were able to multiply in the macrophages in control wells, where they increased from about 130 × 10³ ± 77 × 10³ per 10⁶ cells after infection to 380 × 10³ ± 124 × 10³ per 10⁶ cells at the end of incubation. After 7 days of contact, BM212 completely inhibited the intracellular mycobacteria. The effect was dose dependent, and the MIC was found to be 0.5 μg/ml. From a concentration of 1 μg/ml onwards the inhibition was 100%. Similar results were obtained with RIF at 3 μg/ml. No relevant macrophage loss was detected after 10 days of incubation, both in the control and in the compound-treated cultures. Furthermore, BM212 exerted no inhibition on U937 cell culture replication up to a concentration of 12.5 μg/ml.

The pyrrole derivative BM212 shows some interesting antimicrobial properties: (i) it is strongly inhibitory against both M. tuberculosis and M. avium, which are the two most common mycobacteria causing infection in immunosuppressed patients; and (ii) it also has marked activity against several species of yeasts, including Candida albicans and Cryptococcus neoformans (5). Considering the increased incidence of opportunistic infections caused by candidae and mycobacteria in immunocompromised patients, the development and use of new compounds, which would be active against both these types of microorganisms, is very attractive. Furthermore, BM212 is also highly efficacious against mycobacteria which show resistance to the most common traditional drugs, displaying no cross resistance with them, and it exerts bactericidal activity on intracellular mycobacteria. This fact is very important because mycobacteria can reside for years inside lymphoid cells and macrophages, where they are difficult to get rid of.

In conclusion, BM212, the most potent pyrrole derivative studied so far, can be used as a lead for the preparation of new and more efficacious antimycobacterial drugs. Work is in progress to determine the pharmacokinetic characteristics of the compound in order to evaluate its potential therapeutical value and its mechanism of action.