In Vitro and In Vivo Activity of Oxazolidinone Candidate OTB-658 against Mycobacterium tuberculosis

ABSTRACT

In this work, we assess antituberculosis activity of OTB-658 in vitro and in vivo. In vitro, OTB-658 showed bacteriostatic effectiveness with a lower MIC than linezolid against Mycobacterium tuberculosis. The minimal bactericidal concentrations and time-kill curves for OTB-658 indicated inhibition activity similar to that of linezolid. OTB-658 entered macrophages to inhibit M. tuberculosis growth. OTB-658 had a low mutation frequency (10⁻⁸), which would prevent drug-resistant mutations from emerging in combination regimens. In vivo, OTB-658 reduced CFU counts in the lungs and slightly inhibited bacterial growth in the spleen in the early stage and steady state in acute and chronic murine TB models. These results support the preclinical evaluation of OTB-658 and further clinical trials in China.

TEXT

Tuberculosis (TB) is a major infectious disease globally (1). In particular, multidrug-resistant tuberculosis (MDR-TB) is a severe threat to public health. In 2019, an estimated 3.3% of new TB cases and 18% of previously treated TB cases had MDR-TB or rifampin (RIF)-resistant TB (1). MDR-TB treatment lasts at least 9 to 24 months, with a high probability of adverse effects and a low success rate. To achieve the global goal of ending TB, there is a pressing need to develop new and repurposed compounds as anti-TB agents.

Linezolid (Lzd), the first oxazolidinone approved for clinical use in Gram-positive infections, inhibits cellular proliferation by preventing mitochondrial protein synthesis via binding at the ribosomal 50S subunit (2, 3, 17). Lzd exhibits antimycobacterial activity in vitro against the Mycobacterium tuberculosis complex, including multidrug-resistant Mycobacterium bovis and clinical drug-resistant M. tuberculosis (4, 5). Prospective studies and retrospective analyses have shown that Lzd is effective in treating MDR-TB and extremely drug-resistant TB; thus, Lzd is increasingly used in TB patients (6–12).

However, the main drawback of Lzd is adverse events, including anemia, peripheral neuropathy, and gastrointestinal and hematological reactions, which cause discontinuation of treatment (7, 13). Consequently, there is an urgent need for new oxazolidinone candidates with an improved clinical safety and efficacy profile compared with Lzd.

In our previous work, we synthesized OTB-658, which is a conformationally constrained oxazolidinone candidate, through focused lead optimization, and the compound has an excellent activity, safety, and druggability profile (Fig. 1) (14). In the present work, we evaluate the anti-TB activity of OTB-658 both in vitro and in vivo.

FIG 1.

The structure of compound OTB-658.

RESULTS

MIC determination.

The MICs of OTB-658 and control compounds were shown in Table 1. Isoniazid (INH), RIF, Lzd, and PNU-100480 were used as controls. The MIC value of OTB-658 against M. tuberculosis H37Rv was 0.080 to 0.115 μg/ml, which indicated that OTB-658 was more active than Lzd and PNU-100480. Likewise, OTB-658 was active against 10 drug-susceptible and 30 drug-resistant clinical isolates and was as potent as Lzd and PNU-100480. The MIC values of OTB-658 against 30 drug-resistant clinical isolates of M. tuberculosis were low compared with those of Lzd. No cross-resistance was observed between OTB-658 and INH/RIF in any multidrug-resistant clinical isolate (Table 1).

TABLE 1.

MIC and MBC values of drugs against M. tuberculosis strains

Test compound	M. tuberculosis H37Rv		Drug-susceptible M. tuberculosis clinical isolates (n = 10)		Multidrug-resistant M. tuberculosis clinical isolates (n = 30)
	MIC (μg/ml)	MBC	MIC (μg/ml)	MBC	MIC (μg/ml)	MBC
INH	0.030–0.056	NAa	<0.016–0.449	NA	>1	NA
RIF	0.115	NA	0.027–0.220	NA	>1	NA
PNU-100480	0.121–0.205	NA	0.029–0.115	NA	<0.016–0.207	NA
Lzd	0.331–0.420	>32 MIC	0.095–0.3	>32 MIC	0.036–0.499	>32 MIC
OTB-658	0.080–0.115	>32 MIC	0.008–0.015	>32 MIC	<0.016–0.167	>32 MIC

^aNA, not applicable.

MBC determination.

To understand the properties of OTB-658 against M. tuberculosis H37Rv and six clinical isolates further, the minimal bactericidal concentrations (MBC) was determined. The MBC demonstrated that OTB-658 was bacteriostatic against M. tuberculosis at the MIC to 32× MIC. The growth of bacteria incubated with OTB-658 and Lzd was moderately inhibited. The final CFU counts in both the OTB-658 and Lzd groups were no less than 2.75 log₁₀ CFU/ml, even at 32× MIC. In our study, the bactericidal activity of OTB-658 against clinical isolates did not result in 3-log₁₀ CFU/ml reduction in bacterial cell counts at any concentration. Thus, OTB-658 only inhibited the growth of M. tuberculosis and did not kill it (Table 1).

Time-kill curve analysis.

The time-kill curve analysis of OTB-658 and LZD against M. tuberculosis H37Rv is presented in Fig. 2. OTB-658 exhibited inhibitory effects on H37Rv and two clinical isolates from MIC to 4× MIC. As the OTB-658 concentration increased to 32× MIC, there was no significant CFU reduction when M. tuberculosis was cultured with OTB-658. OTB-658 inhibited the growth of M. tuberculosis in a time-dependent manner. The time-kill curves against two clinical isolates had a similar tendency with those against H37Rv (the curves were not shown here). The results were consistent with the MBC results. OTB-658 showed a time-kill curve similar to that of Lzd.

FIG 2.

Time-kill curves of OTB-658 and Lzd against M. tuberculosis H37Rv.

Antituberculosis activity against intracellular M. tuberculosis.

The intracellular killing activity of OTB-658 was assessed ex vivo after infection of J774A.1 macrophage cells with M. tuberculosis. The infected macrophage cells were exposed to seven drugs and OTB-658 at 0.5, 2, and 5 μg/ml. The intracellular anti-TB activity of OTB-658 was as potent as that of RIF (Table 2). OTB-658 reduced the CFU counts from 5.42 ± 0.13 to 4.97 ± 0.10 at 0.5 μg/ml, to 4.65 ± 0.10 at 2.0 μg/ml, and to 4.53 ± 0.07 at 5.0 μg/ml. OTB-658 showed intracellular anti-TB activity similar to that of RIF; thus, OTB-658 penetrated macrophage cells and inhibited M. tuberculosis H37Rv growth.

TABLE 2.

Intracellular anti-TB activity of agents

Test compound	Activity (log10 CFU/ml) ata:
	5 μg/ml	2 μg/ml	0.5 μg/ml
RIF	4.50 ± 0.06	4.61 ± 0.05	5.15 ± 0.04
INH	3.84 ± 0.09	4.08 ± 0.18	4.36 ± 0.18
SM	4.32 ± 0.24	4.53 ± 0.14	5.14 ± 0.13
EMB	3.87 ± 0.12	4.15 ± 0.11	4.43 ± 0.07
LFX	3.80 ± 0.14	4.84 ± 0.09	5.45 ± 0.04
Lzd	4.39 ± 0.07	4.59 ± 0.06	5.13 ± 0.02
OTB-658	4.53 ± 0.07	4.65 ± 0.10	4.97 ± 0.10

^aActivity of the control was 5.42 ± 0.13 log₁₀ CFU/ml.

Spontaneous mutation frequency.

Spontaneous mutants were resistant to OTB-658 arose at a frequency ranging from 9.09 × 10⁻⁹ to 3.3 × 10⁻⁸ in wild strains of M. tuberculosis when plated on 7H10 solid medium containing 4× MIC OTB-658, which was similar to the frequency for Lzd (1.25 × 10⁻⁹ to 5 × 10⁻⁸).

Antituberculosis activity in an acute murine TB model.

In vivo anti-TB activity of OTB-658 was evaluated in an acute infection murine TB model (Fig. 3, Table 3). OTB-658 exhibited potent activity against M. tuberculosis H37Rv in the early stage of infection. The CFU count in the OTB-658 50 mg/kg group was approximately 3 log₁₀ CFU lower than that in the CMC group (P < 0.001). The CFU count in the OTB-658 50-mg/kg group was significantly lower than that in the Lzd group (3.96 ± 0.37 log₁₀ CFU/lung) (P < 0.001) and was similar to that in the PNU-100480 group (P = 0.287). As the OTB-658 dose increased, a dose-dependent inhibition effect was observed in mice. Remarkably, the activity of OTB-658 at 25 mg/kg was superior to that of Lzd at 50 mg/kg, demonstrating that OTB-658 had better bacteriostatic activity in the early stage of infection in vivo. No significant differences in body weight were observed among the groups (P > 0.05).

FIG 3.

Antituberculosis activity of test compounds in an acute murine model.

TABLE 3.

CFU counts and body weight of mice in an acute infection model

Group	Dose (mg/kg)	Body wt (g)	Log10 CFU
Day 3			2.55 ± 0.17
Day 10			3.91 ± 0.19
CMC		20.85 ± 0.95	5.05 ± 0.32
Lzd	50	20.72 ± 1.00	3.96 ± 0.37
PNU-100480	50	20.18 ± 0.97	2.56 ± 0.24
OTB-658	25	20.61 ± 0.78	3.10 ± 0.24
OTB-658	50	20.34 ± 1.10	2.09 ± 0.30
OTB-658	100	20.71 ± 1.11	1.58 ± 0.85
INH	25	21.27 ± 1.02	0.00

Antituberculosis activity in chronic murine TB model.

We evaluated the in vivo activity of OTB-658 in a chronic murine TB model. After 4 weeks of infection, the initial CFU counts reached 5.52 ± 0.15 log₁₀ CFU in the lungs. The CFU counts in the lungs steadily declined during treatment. The bacterial load in the mice in the 25-, 50-, and 100-mg/kg OTB-658 groups reduced by approximately 1.5, 2.5, and 3 log₁₀ CFU/lung after 8 weeks of treatment compared with the Lzd and CMC groups, demonstrating significant activity in vivo (P < 0.001) (Fig. 4).

FIG 4.

Antituberculosis activity in the lungs in a chronic murine model.

In the spleen, the CFU counts in all groups except the RIF group increased rapidly during the first 4 weeks. Subsequently, the CFU counts in three OTB-658 groups reduced slightly. The antituberculosis efficacy appeared to be dose dependent. OTB-658 was much more potent than Lzd at 50 and 100 mg/kg against M. tuberculosis (P < 0.001). No CFU count reduction was observed in the Lzd group (Fig. 5).

FIG 5.

Antituberculosis activity in the spleen in a chronic murine model.

DISCUSSION

In 2000, Lzd was the first oxazolidinone drug used in clinical treatment of TB (15). Before that, Lzd had already been used clinically for treating Gram‐positive infections (16, 17). Lzd is also potent against MDR-TB isolates in vivo (4, 18, 19). Until 2018, WHO reclassified Lzd as a group A drug for MDR-TB (20). Several new oxazolidinones, including AZD5847 and PNU-100480, have been discovered and their antibacterial activity assessed (21–26). We modified the structure of Lzd and introduced various substituents to increase the in vitro and in vivo activity against M. tuberculosis. Compounds with a tricyclic fused benzoxazinyl-oxazolidinone scaffold have potent antibacterial activity (27–29), and thiomorpholine substitution and amino substitution can also increase activity (14). We recommended that the anti-TB activity of thiomorpholine-substituted compound OTB-658 should be evaluated in vitro and in vivo (14).

The MIC value of OTB-658 against M. tuberculosis showed anti-TB activity 2- to 4-fold more potent than those of Lzd and PNU-100480 in vitro. OTB-658 also exhibited excellent in vitro activity against multiple clinical isolates of M. tuberculosis. Similar to Lzd and PNU-100480, OTB-658 showed no cross-resistance with INH and RIF (24, 30). In addition, OTB-658 is bacteriostatic rather than bactericidal according to the MBC value in our study. It has been reported that oxazolidinones are bacteriostatic, with an MBC/MIC ratio of >16 (23, 31). Time-kill curve analysis of OTB-658 confirmed this observation. OTB-658 did not significantly decrease the CFU count, even at high concentration. The decrease in CFU count also showed that OTB-658 is a bacteriostatic agent, similar to Lzd, rather than a bactericidal agent. The kinetic curve patterns of OTB-658, which are similar to that of Lzd, demonstrated that inhibitory activity depends on exposure time to OTB-658 in culture.

Because M. tuberculosis is trapped and embedded in multiple layers of immune cells and necrotic tissue, the penetration efficacy of therapeutic agents is crucial (32). In our previous study, OTB-658 had similar inhibitory effect with Lzd against M. tuberculosis H37Rv in macrophages (33). The data in these two studies were consistent. As Lzd, OTB-658 is able to pass through the walls of macrophage cells to inhibit the growth of M. tuberculosis.

The mutant frequencies of OTB-658 and Lzd were 10⁻⁸ to 10⁻⁹, which were lower than those of INH and RIF (34, 35). Antituberculosis drugs with low spontaneous mutant frequencies do not increase resistant mutagenesis greatly. The incidence of resistance to INH is as high as 20% to 30%, compared with Lzd resistance of 1.9% (4 of 210) in clinical strains (35, 36). It is difficult to induce Lzd-resistant mutants in vitro when culturing resistant strains in the hollow fiber system model for TB and on Lzd-containing solid medium (36–38). Thus, anti-TB efficacy and prevention of resistance should be further evaluated in mice.

Although neither OTB-658 nor Lzd could kill bacteria in vitro, we found that OTB-658 exhibited superior efficiency to Lzd in vivo. Similar findings have been reported for other new oxazolidinones (26), and the activity of other new oxazolidinones has been reported in an acute infection model (39, 40). It has been shown that OTB-658 has in vivo efficacy superior to that of Lzd and PNU-100480 at the same dose in an acute model (14). In other words, the growth of M. tuberculosis could be inhibited or even reduced by OTB-658 without being suppressed by the host’s immune system, because the immune reaction of mice is not fully established in the early stage of the disease. The time-kill curve in vitro and the acute model in vivo produced inconsistent results. This inconsistency may explain the metabolism progress of OTB-658 between in vitro and in vivo. Previous studies have found that two active metabolites of OTB-658 could be converted into two active metabolites in monkeys, and these two active metabolites could also show anti-TB activity against M. tuberculosis (15, 40). These metabolites may explain why OTB-658 exhibits bactericidal activity in vivo.

In the chronic infection model, we observed greater CFU count reduction in the two OTB-658 groups than in the Lzd group, which is consistent with the results in our previous study, showing that OTB-658 was better at sterilizing stationary-phase-growth M. tuberculosis (14). This result can be explained by the favorable pharmacokinetic profiles of new oxazolidinone compounds. According to an ADME study of OTB-658 in vivo, OTB-658 exhibited a long half-life in human hepatocytes of >500 min and >14.7 h in mice and had excellent areas under the concentration-time curve (AUCs) (14). In addition, OTB-658 had a short time to reach the maximum concentration in mice, which indicates that OTB-658 could be absorbed rapidly into the bloodstream and then maintain a steady concentration (14). The long elimination half-life would help to reduce the dosing frequency. In addition, time the concentration remains above the MIC and the AUC/MIC of Lzd in vivo were consistently correlated with efficacy when bacterial multiplication was constrained by host immunity or combination with pretomanid (39). As an Lzd analogue, OTB-658 may have pharmacokinetic and pharmacodynamic properties similar to those of Lzd. The favorable pharmacokinetic profile of OTB-658 may improve its in vivo efficacy compared with Lzd.

OTB-658 was active against slowly replicating M. tuberculosis, with even better sterilizing efficacy than RIF in the OTB-658 50- and 100-mg/kg groups. RIF is critical for shortening the treatment duration for patients with drug-susceptible TB to 6 months when combined with pyrazinamide in the first 2 months (42). Superior inhibition of persistent M. tuberculosis would advance the development of short-course treatments for TB (43).

Adverse drug reactions, including thrombocytopenia, marrow suppression, and optic and peripheral neuropathy, are the main obstacles for clinical use of Lzd (44). Lzd can interrupt mitochondrial protein synthesis and inhibit monoamine oxidase (14, 45). Higher mean Lzd trough concentrations were associated with lower mitochondrial function levels (46, 47). In addition, the duration of Lzd-containing therapy is a risk factor for anemia, especially for patients who receive Lzd for >15 days (46). Patients are given Lzd orally for at least 6 months, regardless of treatment duration (20). In contrast, OTB-658 has good performance with low toxicity and high efficacy. OTB-658 showed lower inhibition of mitochondrial protein synthesis and monoamine oxidase than Lzd and sutezolid (14). Therefore, OTB-658 is expected to have a favorable safety profile during clinical use.

In 2020, OTB-658 received approval for conducting clinical trials by National Medical Products Administration in China. It expects a phase 1 clinical trial to determine treatment safety on human. Meanwhile, further preclinical research on OTB-658 would be performed continuously, including adding OTB-658 to anti-TB regimens. Lzd-containing combinations, such as Nix-TB regimen, showed favorable outcomes against M. tuberculosis (12, 16, 47, 49–52). The use of OTB-658 in combinations in order to improved efficacy and shorten the treatment duration should be further investigated.

MATERIALS AND METHODS

Drugs and compounds.

INH (≥99%; batch no. MKBW9046V), RIF (≥97%; batch no. BCBP4553V), ethambutol (EMB; ≥99%; batch no. 071M0201V), and levofloxacin (LFX; ≥98%; batch no. BCBF7004V) were purchased from Sigma-Aldrich Co., Ltd. (Shanghai, China). Streptomycin (SM; batch no. P09615) was purchased from Adamas-Beta Inc. (Shanghai, China). Lzd (>97%; batch no. WG0042607-170220001) was purchased from Ark Pharm, Inc. (Arlington Heights, IL, USA). PNU-100480 (batch no. BF00174457) was provided by the TB Alliance (New York, USA). OTB-658 (99.52%; batch no. 20190110) was provided by the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China).

Strains.

M. tuberculosis H37Rv was obtained from the American Type Culture Collection (no. 27294). Clinical strains of M. tuberculosis were obtained from the National Clinical Laboratory on Tuberculosis in the Beijing Tuberculosis and Thoracic Tumor Research Institute. M. tuberculosis H37Rv and the clinical strains were cultured in Middlebrook 7H9 broth (Difco) with 0.05% Tween 80 (Sigma-Aldrich) and 10% (vol/vol) oleic acid albumin dextrose catalase (OADC; Becton-Dickinson and Company, Franklin Lakes, NJ, USA). After 2 weeks of incubation at 37°C and 5% CO₂, the logarithmic-phase bacterial culture was used as the inoculum for further experiments. Bacteria were quantified by measuring optical density at 570 nm (OD₅₇₀) using a conversion of 1 × 10⁸ CFU/ml for an OD₅₇₀ of 1.

MIC.

The microplate alamarBlue assay (MABA) method was used to determine the MICs of OTB-658 and Lzd against the M. tuberculosis H37Rv strain and 40 susceptible and drug-resistant M. tuberculosis clinical isolates. INH, RIF, Lzd, and PNU-100480 were selected as positive controls. The details of the procedure have been described previously (53). Serial 2-fold dilutions of the test compounds (100 μl each) were added to each well of a 96-well black-frame microplate, followed by the bacterial suspension (100 μl, 2 × 10⁵ CFU/ml). The final concentrations of OTB-658, PNU-100480, INH, and RIF ranged from 1.0 to 0.0156 mg/liter, whereas that of Lzd ranged from 2.0 to 0.0313 mg/liter. Control wells contained only the medium and culture controls with no test compounds. The plate was incubated at 37°C and 5% CO₂ for 7 days. A mixture of alamarBlue (Bio-Rad) and 20% Tween 80 (32.5 μl) was added to each culture well, and the plate was reincubated for another 16 to 24 h. The fluorescence of each well was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm (Infinite M200; Tecan). The MIC is defined as the minimum concentration of a test drug at which there is a reduction in fluorescence of ≥90% compared with the mean fluorescence of replicate drug-free controls.

Minimal bactericidal concentration.

The CFU-based enumeration method was used to determine minimal bactericidal concentrations (MBCs) against M. tuberculosis H37Rv and six M. tuberculosis clinical isolates in 96-well culture plates cultured after 7 days of incubation with the compounds. According to the Clinical and Laboratory Standards Institute guidelines, the MBC value was determined by 99.9% killing of the final inoculum (54). The initial number of CFU was calculated on the same day as when the culture was incubated in 96-well microplates. The culture concentration ranges were from MIC to 32× MIC for OTB-658 and Lzd. After 7 days of incubation, culture aliquots from each well were plated on blank 7H10 agar (Difco). CFU on all plates were counted after 28 days in incubation. The MBC was defined as the lowest concentration of the drugs that caused at least a 3-log₁₀ decrease in the number of CFU compared with the initial number of CFU.

Time-kill curve.

Time-kill curve analyses were performed by culturing M. tuberculosis in 7H10 solid agar medium. Bacterial culture (2 × 10⁵ CFU/ml) was added to wells in the presence of OTB-658 and Lzd concentrations in doubling dilutions ranging from 0.5× MIC to 16× MIC in 24-well plates. For all strains, the MICs were determined previously. M. tuberculosis simultaneously cultured in drug-free 7H9 broth served as the growth control for each strain. At 0, 3, 7, 10, and 14 days, 10-fold serial dilutions of culture suspension were extracted and plated on 7H10 solid medium. After 28 days of incubation, CFU counts on all the plates were recorded. The killing rate of M. tuberculosis by each drug was determined by plotting log₁₀ CFU/ml versus culture time.

Activity in J774A.1 macrophage cell.

M. tuberculosis H37Rv was cultivated in 7H9 broth for 2 to 3 weeks so that it was in the exponential phase. A J774A.1 macrophage cell infection model was established after coincubation with M. tuberculosis H37Rv for 4 h. The infected J774A.1 macrophages were washed several times with phosphate-buffered saline (PBS) to remove residual M. tuberculosis H37Rv from the liquid medium. The candidate compounds and control drugs were added to the medium, and the J774A.1 macrophages were reincubated for another 72 h. The infected J774A.1 macrophages were washed with PBS to remove compound-containing medium, and then the macrophage cells were treated with SDS lysis buffer (Sigma-Aldrich). The original culture and 10-fold serial dilutions of culture suspension were extracted and plated on 7H10 solid medium for 3 to 4 weeks of incubation.

Spontaneous mutant frequency.

Four replicate cultures of M. tuberculosis H37Rv were prepared in 600-ml flasks, each containing Middlebrook 7H9 broth (10 ml) with glycerol and OADC supplementation at an initial M. tuberculosis density of 1 × 10⁴ CFU/ml, and the cultures were monitored for the presence of preexisting resistant bacteria by plating on 7H10 solid medium containing the test compound at 4× MIC. The cultures were then incubated for another 3 weeks with shaking until an OD₅₇₀ of 1.0 was obtained. From each M. tuberculosis H37Rv culture, 100-μl aliquots of undiluted and 1:10 diluted suspensions were plated on 10 plates of 7H10 agar with and without 4× MIC OTB-658 and Lzd. Individual mutation frequencies were calculated for each of the four cultures, and the median value was selected as representative.

Establishment of murine TB infection model.

Female BALB/c mice, weighing 18 to 20 g (in-house breeding), were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. Five mice were housed in each cage with sterile shaving for bedding. The temperature was maintained at 21 ± 2°C, and the relative humidity level was 55% ± 15% with a 12:12 h dark-light cycle. The mice were allowed free access to water and food. Log-phase M. tuberculosis H37Rv strains were suspended evenly with 5 × 10⁶ CFU/ml for aerosol infection (099C A4224 inhalation exposure system; Glas-Col). For acute and chronic models, the baseline bacterial load could be determined after 10 days and 6 weeks of aerosol infection, respectively. The mice were randomly assigned to treatment groups.

Treatment for acute and chronic TB infection murine models.

Animal experiments were approved by the Animal Ethics Committee of Beijing Chest Hospital, Capital Medical University, and all animal procedures were approved by the Animal Care and Use Committee (permit no. 10923).

Efficacy was assessed by CFU counts in the lungs and spleen in mice. Drugs and the candidate compound were prepared in 0.5% (wt/vol) carboxymethylcellulose (CMC). CMC served as the negative-control vehicle. Treatment was performed five times per week. For the acute model, treatment lasted for 3 weeks. The positive controls were INH, 25 mg/kg, Lzd, 50 mg/kg, and PNU-100480, 50 mg/kg, and OTB-658 was administered at doses of 25, 50, and 100 mg/kg. After treatment, all mice were sacrificed, and the lungs were homogenized in PBS. For the chronic model, treatment lasted for 8 weeks. The positive controls were RIF, 10 mg/kg, and Lzd, 50 mg/kg. The other three tested groups were administered candidate OTB-658 at low, medium, and high doses (25, 50, and 100 mg/kg, respectively, per os). Five mice in each group were sacrificed by cervical dislocation after 2, 4, and 8 weeks of drug administration, and the lungs and spleen were homogenized. Tenfold serial dilutions of the homogenate were plated on 7H10 agar enriched with 10% OADC and incubated at 37°C and 5% CO₂ for 3 to 4 weeks.

Statistical analysis.

CFU counts (X) of each plate were calculated and log-transformed as (X + 1) before analysis. The groups’ means were compared by two-way analysis of variance. The results are expressed as means ± standard deviations. Statistical analysis was performed by SPSS 25.0 (IBM).