Efficacy of Replacing Linezolid with OTB-658 in Anti-Tuberculosis Regimens in Murine Models
ABSTRACT
Linezolid (LZD) was the first oxazolidinone approved for treating drug-resistant tuberculosis. A newly approved regimen combining LZD with bedaquiline (BDQ) and pretomanid (PMD) (BPaL regimen) is the first 6-month oral regimen that is effective against multidrug- and extensively drug-resistant tuberculosis. However, LZD toxicity, primarily due to mitochondrial protein synthesis inhibition, may undermine the efficacy of LZD regimens, and oxazolidinones with higher efficacy and lower toxicity during prolonged administration are needed. OTB-658 is an oxazolidinone anti-TB candidate derived from LZD that could replace LZD in TB treatment. We previously found that OTB-658 had better anti-TB activity and safety than LZD in vitro and in vivo. In the present work, two murine TB models were used to evaluate replacing LZD with OTB-658 in LZD-containing regimens. In the C3HeB/FeJ murine model, replacing 100 mg/kg LZD with 50 mg/kg OTB-658 in the BDQ + PMD backbone significantly reduced lung and spleen CFU counts (P < 0.05), and there were few relapses at 8 weeks of treatment. Replacing 100 mg/kg LZD with 50 or 100 mg/kg OTB-658 in the pyrifazimine (previously called TBI-166) + BDQ backbone did not change the anti-TB efficacy and relapse rate. In BALB/c mice, replacing 100 mg/kg LZD with 100 mg/kg OTB-658 in the TBI-166 + BDQ backbone resulted in no culture-positive lungs at 4 and 8 weeks of treatment, and there were no significant differences in relapses rate between the groups. In conclusion, OTB-658 is a promising clinical candidate that could replace LZD in the BPaL or TBI-166 + BDQ + LZD regimens and should be studied further in clinical trials.
INTRODUCTION
Tuberculosis (TB) remains a serious global health threat. The World Health Organization (WHO) estimated that worldwide in 2020, there were nearly 2 billion people with latent TB infection, about 9.9 million people suffered from the disease, and nearly 500,000 new cases of multidrug-resistant tuberculosis (MDR-TB) occurred annually, with an estimated treatment success rate of 59% (1). The WHO recommends a new shorter-course treatment regimen (known as Bangladesh regimen) for MDR-TB that takes at least 9 to 12 months in 2016 (2); however, widespread use of the regimen has been limited by its narrow scope of treatment and toxic side effects (3). Therefore, new drugs and regimens with high efficacy and low toxicity to shorten and simplify TB treatment are urgently needed.
Linezolid (LZD), the first oxazolidinone approved for clinical use in Gram-positive infections, is effective in treating MDR-TB and extremely drug-resistant TB; thus, LZD is increasingly used in drug-resistant TB patients (4). However, the main drawback of LZD is adverse events, including myelosuppression and peripheral neuropathy, which are thought to be caused by inhibition of mitochondrial protein synthesis and monoamine oxidase (5–9), causing discontinuation of treatment (10, 11). Therefore, further optimization of oxazolidinone derivatives is required to maintain the anti-TB activity but reduce the toxicity. The study of the structure-activity relationship of these compounds resulted in derivative OTB-658 (Fig. 1), which has potent anti-TB activity with a favorable safety profile (12). OTB-658 showed better efficacy than LZD in vitro and in a murine model of TB infection (13), and it showed lower bone marrow suppression than LZD in a dose toxicity study of OTB-658 in rats, which is the main disadvantage of oxazolidinones (12, 14). In 2020, OTB-658 was approved for clinical trials by the National Medical Products Administration in China, and a phase 1 clinical trial to determine its safety in humans is ongoing.
FIG 1
The BPaL regimen (Nix-TB regimen), consisting of Bedaquiline (BDQ), pretomanid (PMD), and LZD, is expected to shorten the duration of treatment for drug-resistant TB to 6 months (15, 16). In addition, the WHO issued a rapid communication on the updated guidance that programmatic use of the 6-month BPaL(M) regimen for the treatment of drug-resistant tuberculosis in 2022. However, LZD has a narrow therapeutic window when used at a high daily dose for long periods in the BPaL regimen. Our previous study found that a new LZD-containing regimen, TBI-166 + BDQ + LZD regimen, had the highest bactericidal activity of all TBI-166-containing regimens in C3HeB/FeJNju mice and was superior to the standard first-line isoniazid + rifampin + pyrazinamide regimen (17).
Because OTB-658 has higher bactericidal activity in the mouse model and a better preclinical safety profile than LZD, in this study, two murine models of TB were used to evaluate the effect of replacing LZD with OTB-658 on the bactericidal and sterilizing activity of the BPaL and TBI-166 + BDQ + LZD regimens.
RESULTS
Experiment 1: anti-TB activity of regimens in which LZD was replaced with OTB-658 in C3HeB/FeJ mice.
In this experiment, C3HeB/FeJ mice were used to compare the effect on anti-TB activity of replacing 100 mg/kg LZD with 50 mg/kg OTB-658 (OTB-658(50)) in the BPaL (BpaL(100)) and TBI-166 + BDQ + LZD (LZD(100)) regimens, or with 100 mg/kg OTB-658 (OTB-658(100)) in the TBI-166 + BDQ + LZD(100) regimen.
The CFU counts for the lungs and spleen in the C3HeB/FeJ mice at 8 weeks of treatment are shown in Table 1. Treatment (D0) began 21 days after infection, when the mean CFU count was 4.92 log10 CFU in the lungs and 2.03 log10 CFU in the spleen. The mean bacterial burden of the untreated mice remained high throughout the experiment, with a mean CFU count of 8.22 log10 CFU in the lungs and 4.47 log10 CFU in the spleen after 8 weeks, whereas all drug regimens were significantly bactericidal (P < 0.001). After 8 weeks of administration, 60% of the mice (3/5) in the BDQ + PMD + OTB-658(50) group had culture-positive (this meant there was viable bacteria cultured on 7H10 plates) lung tissue, and the negative conversion rate was slightly higher than for the BpaL(100) regimen. Treatment with BDQ + PMD + OTB-658(50) reduced the bacterial burden by more than 7.50 log10 CFU in the lungs and 3.89 log10 CFU in the spleen compared to the untreated control. These were greater reductions than for the BpaL(100) regimen, which reduced the bacterial burden by 7.15 log10 CFU in the lungs and 3.27 log10 CFU in the spleen, and the differences were statistically significant (P < 0.05). Based on the TBI-166 + BDQ + LZD regimen, we also compared the anti-TB activity of the regimens in which 100 mg/kg LZD was replaced with 50 or 100 mg/kg OTB-658. Only 20% of the mice (1/5) had culture-positive lung tissue in the TBI-166 + BDQ + LZD(100) regimen, which indicated that the TBI-166 + BDQ + LZD(100) regimen had better anti-TB activity than the TBI-166 + BDQ + OTB-658 regimens. In addition, except for TBI-166 + BDQ + OTB-658(50), the anti-TB activities of all other regimens were slightly better than that of the BpaL(100) regimen (Fig. 2).
FIG 2
TABLE 1
| Mean log10 CFU counta (positive lung or spleen culture) | Proportion of mice relapsed after treatment (percent relapse in group)c | ||
|---|---|---|---|
| Groupsb | D0 | W8 | W8 (+W12) |
| Lungs | |||
| Untreated | 4.92 ± 0.18 (3/3) | 8.22 ± 0.21 (5/5) | |
| BpaL(100) | 1.07 ± 0.44 (5/5) | 0/5 (0%) | |
| BDQ + PMD + OTB-658(50) | 0.72 ± 0.42 (3/5) | 0/5 (0%) | |
| TBI-166 + BDQ + LZD(100) | 0.95 (1/5) | 1/5 (20%) | |
| TBI-166 + BDQ + OTB-658(50) | 0.65 ± 0.32 (5/5) | 5/5 (100%)* | |
| TBI-166 + BDQ + OTB-658(100) | 0.53 ± 0.29 (4/5) | 2/5 (40%) | |
| Spleen | |||
| Untreated | 2.03 ± 0.89 (5/5) | 4.47 ± 0.17 (5/5) | |
| BpaL(100) | 1.20 ± 0.46 (4/5) | 0/5 (0%) | |
| BDQ + PMD + OTB-658(50) | 0.58 ± 0.34 (3/5) | 0/5 (0%) | |
| TBI-166 + BDQ + LZD(100) | 0.30 (2/5) | 0/5 (0%) | |
| TBI-166 + BDQ + OTB-658(50) | 0.69 ± 0.12 (2/5) | 4/5 (80%) | |
| TBI-166 + BDQ + OTB-658(100) | 0.53 ± 0.45 (4/5) | 3/5 (60%) | |
a
Time points are shown as days (D0) or 8 weeks of treatment. The start of the treatment (D0) began 21 days after infection. Values are means ± standard deviation, n = 5.
b
Drugs (abbreviations) and doses: bedaquiline (BDQ), 25 mg/kg; pretomanid (PMD), 100 mg/kg; linezolid (LZD), 100 mg/kg; pyrifazimine (TBI-166), 20 mg/kg; OTB-658(50), 50 mg/kg; OTB-658(100), 100 mg/kg; BpaL(100), BDQ + PMD + LZD(100).
c
Mouse lung and spleen homogenates were cultured on 7H10 plates containing activated carbon at the end of 12 weeks following the discontinuation of the 8 week treatment regimen (W8 + W12). *, P < 0.05 (compared with TBI-166 + BDQ + LZD(100) group). n = 5.
Next, we assessed the relapse rate (the proportion of mice lung relapsing after stopping treatment) by taking five mice each from all drug administration groups and discontinuing the treatment for 12 weeks after 8 weeks of treatment (Table 1). In the five mice in the BpaL(100) and BDQ + PMD + OTB-658(50) groups, there were no culture-positive relapses in mouse lung homogenates. Treatment with the TBI-166 + BDQ + LZD(100) regimen for 8 weeks resulted in the relapse of one (20%) of the five mice. In contrast, five mice in the TBI-166 + BDQ + OTB-658(50) group relapsed and the difference was statistically significant (P < 0.05). Two (40%) of the five mice in the TBI-166 + BDQ + OTB-658(100) group relapsed, although there was no statistically significant difference between the TBI-166 + BDQ + LZD(100) group and the TBI-166 + BDQ + OTB-658(100) group.
Experiment 2: sterilizing activity of the regimens TBI-166 + BDQ + LZD(100) and TBI-166 + BDQ + OTB-658(100) in BALB/c mice.
To further compare the sterilization activities between the TBI-166 + BDQ + OTB-658(100) regimen and TBI-166 + BDQ + LZD(100) regimen, the bacterial activities of the BpaL(100), TBI-166 + BDQ + LZD(100) and TBI-166 + BDQ + OTB-658(100) regimens were observed in a BALB/c murine TB model over 8 weeks. Relapse was also assessed from the lungs at 12 weeks after completion of 4 and 8 weeks of treatment in BALB/c mice (Table 2).
TABLE 2
| Mean log10 CFU count | Proportion of mice relapsed after treatment (percent relapse in group)a | ||||
|---|---|---|---|---|---|
| Groupsc | D0 | W4 | W8 | W4 (+W12) | W8 (+W12) |
| Lungs | |||||
| Untreated | 3.62 ± 0.30 (5/5) | ||||
| BpaL(100) | Negative | Negative | 9/13 (69.23%)b | 0/15 (0%) | |
| TBI-166 + BDQ + LZD(100) | Negative | Negative | 5/15 (33.33%) | 2/14 (14.29%)b | |
| TBI-166 + BDQ + OTB-658(100) | Negative | Negative | 9/15 (60%) | 1/15 (6.67%) | |
| Spleen | |||||
| BpaL(100) | Negative | Negative | 2/13 (15.38%)b | 0/15 (0%) | |
| TBI-166 + BDQ + LZD(100) | Negative | Negative | 4/15 (26.67%) | 0/14 (0%) | |
| TBI-166 + BDQ + OTB-658(100) | Negative | Negative | 3/15 (20%) | 0/15 (0%) | |
a
Time points are shown as days (D0) or weeks (W4 or W8) of treatment. The start of the treatment (D0) began 14 days after infection. Mouse lung and spleen homogenates were cultured on 7H10 plates containing activated carbon at the end of 12 weeks following the discontinuation of the 4 week (W4 + W12) or 8 week (W8 + W12) treatment regimens. ns, not significant (compared with BpaL(100)). n = 15.
b
One mouse in the TBI-166 + BDQ + LZD(100) group and two mice in the BpaL(100) group died due to gavage accidents.
c
Drugs (abbreviations) and doses: bedaquiline (BDQ), 25 mg/kg; pretomanid (PMD), 100 mg/kg; linezolid (LZD), 100 mg/kg; pyrifazimine (TBI-166), 20 mg/kg; OTB-658(100), 100 mg/kg; BpaL(100), BDQ + PMD + LZD(100).
Treatment (D0) began 14 days after infection, when the mean CFU count was 3.62 log10 CFU in the lungs. After 4 and 8 weeks of treatment, all regimens showed high bactericidal activities with no culture-positive lungs and spleen. Based on the lung culture, after 12 weeks following the discontinuation of 4 weeks of treatment with the TBI-166 + BDQ + LZD(100), TBI-166 + BDQ + OTB-658(100), and BpaL(100) regimens, relapse occurred in five (33.33%) of 15 mice, nine (60%) of 15 mice, and nine (69.23%) of 13 mice, respectively. Prolonging the treatment decreased the relapse rate of mice in each drug administration group significantly. After 12 weeks following the discontinuation of 8 weeks of treatment with the TBI-166 + BDQ + LZD(100) and TBI-166 + BDQ + OTB-658(100), relapse occurred in two (14.29%) of 14 mice, one (6.67%) of 15 mice, respectively, and none of the BpaL(100) mice relapsed after 8 weeks of treatment. There were no statistically significant differences in the relapse rates across all drug administration groups, either after 4 or 8 weeks of treatment.
Experiment 3: interactions of the drug combinations TBI-166 + LZD and TBI-166 + OTB-658 in BALB/c mice.
In experiment 1, the anti-TB activities of the TBI-166 + BDQ + OTB-658 regimens appeared to be inferior to that of the TBI-166 + BDQ + LZD regimen. In the relapse experiment at 12 weeks following the discontinuation of 8 weeks treatment with 50 and 100 mg/kg OTB-658 added to the TBI-166 + BDQ backbone, the relapse rate of mice was higher than that of the TBI-166 + BDQ + LZD regimen. Therefore, we speculated whether TBI-166 combined with OTB-658 would produce antagonism in the treatment. BALB/c mice were used to evaluate the interaction of the drug combinations of TBI-166 + LZD and TBI-166 + OTB-658.
The CFU count for the lungs in the BALB/c mice at 4 and 8 weeks treatment is shown in Fig. 2. Treatment (D0) began 10 days after infection, when the mean CFU count was 5.00 log10 CFU in the lungs. At 4 and 8 weeks of administration, the anti-TB activity of the OTB-658 monotherapy group was significantly better than that of the LZD group, which was consistent with the previous findings (13). Combining OTB-658 with TBI-166 resulted in a CFU count for the lungs that was close to that of the OTB-658 group, regardless of whether the combination was administered for 4 or 8 weeks, indicating that TBI-166 did not enhance the anti-TB activity of OTB-658. However, combining LZD with TBI-166 reduced the mean lung CFU count by at least 1 log10 CFU compared with LZD alone at 4 and 8 weeks of administration, and there was a statistically significant difference between the LZD and LZD + TBI-166 groups. Therefore, TBI-166 increased the anti-TB activity of LZD (Fig. 3).
FIG 3
DISCUSSION
There is an urgent need for new drugs and regimens for treating MDR-TB and extremely drug-resistant TB. The first oxazolidinone clinically available was LZD, discovered in 1996 and approved in 2000 for clinical use by the U.S. Food and Drug Administration (18). As a core drug for treating drug-resistant TB, it has been listed by the WHO as a group A drug for treating drug-resistant TB since 2018. However, LZD has a narrow therapeutic window, which leads to bone marrow toxicity and neurotoxicity with high daily doses and long-term use (5). Many efforts have been made to discover the new oxazolidinones with better antibacterial efficacy and safety. Based on the good performance of OTB-658 in oxazolidinone analogue screening, we studied its anti-TB activity further. OTB-658 showed more potent anti-TB activity than LZD in vitro and in vivo (13), and it had good performance with high efficacy and low toxicity and lower inhibition of monoamine oxidase and mitochondrial protein synthesis than LZD and sutezolid (12). A phase 1 clinical trial to determine safety in humans is ongoing. Further preclinical research on OTB-658 is being performed, including replacing LZD with OTB-658 in anti-TB regimens.
Previously, we found that TBI-166 + BDQ + LZD had the highest anti-TB activity of all TBI-166-containing regimens in C3HeB/FeJNju mice (17). In the present experiments, we used two murine models of TB to assess the effects of replacing LZD with OTB-658 on the activity of the TBI-166 + BDQ + LZD regimen in experiments 1 and 2. In addition, because the LZD-containing BPaL regimen is important as a candidate regimen for treating drug-resistant TB, we also used the C3HeB/FeJ murine model of TB to compare the anti-TB activity of replacing LZD with OTB-658 in experiment 1. Replacing 100 mg/kg LZD with 50 mg/kg OTB-658 in the BDQ + PMD backbone significantly reduced the lungs and spleen CFU counts (P < 0.05) and for both regimens, there were almost no relapses at 8 weeks of treatment. Furthermore, replacing 100 mg/kg LZD in the TBI-166 + BDQ backbone with 100 mg/kg OTB-658 maintained the anti-TB efficacy and relapse rate in experiment 1. These results indicated that OTB-658 could be used instead of LZD in the regimens for TB treatment. To confirm this hypothesis, we used the BALB/c murine model of TB to compare the sterilizing activity of the TBI-166 + BDQ + LZD(100) and TBI-166 + BDQ + OTB-658(100) regimens in experiment 2. All regimens showed high bactericidal activities with no positive lung and spleen tissue after 4 and 8 weeks treatment. After 12 weeks, following the discontinuation of 8 weeks of treatment with the TBI-166 + BDQ + LZD(100) and TBI-166 + BDQ + OTB-658(100) regimens, there was no statistically significant difference in relapse rates between the TBI-166 + BDQ + LZD(100) and TBI-166 + BDQ + OTB-658(100) groups.
Replacing LZD with OTB-658 in the BPaL regimen produced stronger anti-TB activity, and even for OTB-658 at 50 mg/kg, the anti-TB activity was still stronger than that of LZD at 100 mg/kg. In contrast, replacing LZD with OTB-658 at 100 mg/kg in the TBI-166 + BDQ + LZD regimen, which had only maintained the anti-TB efficacy and relapse rate in experiment 1. We speculated that the role of LZD in the two regimens was different. In the BPaL regimen, BDQ showed the main bactericidal activity, and the role of LZD was only additional effect on BDQ and PMD when OTB-658 which had higher activity than LZD was substituted, the bactericidal activity of the original regimen was greatly enhanced. However, in the TBI-166 + BDQ + LZD(100) regimen, LZD played an important sterilization role in the regimen besides BDQ. In experiment 3, it showed that the combination of TBI-166 and LZD had synergistic bactericidal activity, so the combined bactericidal activity of the two drugs with BDQ was greatly enhanced. TBI-166 had no interaction with OTB-658; therefore, the bactericidal activity of TBI-166 + BDQ + LZD(100) was stronger than that of TBI-166 + BDQ + OTB-658(100), even if OTB-658 was used to replace LZD. We previously found that TBI-166 combined with BDQ significantly enhanced the anti-TB activity compared with the individual drugs, and that TBI-166 and BDQ would accumulate to varying degrees in tissues (19, 20). This observation also explained why in experiment 1 we found that even if mice had live bacteria in their lungs and spleen after 8 weeks of treatment, in the relapse observation after discontinuing treatment for 12 weeks, the organs were no culture-positive. We speculate that when the treatment was discontinued for 12 weeks, TBI-166 and BDQ in tissues were redistributed to the target organs and continued to exert bactericidal activity.
C3HeB/FeJ mice are increasingly used in TB drug development because they can produce caseous necrotic lung lesions, including cavities, which are more similar to the pathological features of human TB (21–23). BALB/c mice only have cellular granulomas after infection with M. tuberculosis, whereas C3HeB/FeJ mice have necrotic lesions. The distribution of drugs across the lesion into the caseum is altered, and there are different microenvironments in the caseum, resulting in reduced drug activity, which has a profound impact on drug efficacy (24–26). For example, in previous studies, reduced diffusion of BDQ was observed in the caseum in necrotic lung lesions compared with adjacent cell regions, and the activity of PZA was reduced in these areas with near-neutral pH (25, 26). In contrast, PMD diffuses well in the caseum and is active under hypoxic conditions (27). Therefore, in this study we used two murine models to assess the anti-TB activity of the new regimens, we found that the BpaL(100), BDQ + PMD + OTB-658(50), TBI-166 + BDQ + LZD(100), and TBI-166 + BDQ + OTB-658(100) regimens all showed strong anti-TB activity, and the TBI-166 + BDQ + LZD(100) and TBI-166 + BDQ + OTB-658(100) regimens had similar relapse rates after 12 weeks following the discontinuation of 8 weeks of treatment in both animal models.
Our study has several limitations. First, the short treatment time and high amount of bacteria in experiment 1 meant that we could not comprehensively compare the efficacy and relapse rates of the regimens from one study alone. In experiment 1, in C3HeB/FeJ mice, due to the high amount of bacteria on the day of treatment and short treatment time, the organs of the mice were culture-positive after 8 weeks of administration, which may have affected the relapse. Second, in experiment 1, we performed a preliminary study of the antibacterial and sterilizing activities of each regimen, so we used only five mice per group to assess relapse, which may not have been a true reflection of the relapse rate. Therefore, in experiment 2 we increased the number of mice per group. Third, the study was performed with the laboratory H37Rv strain only. It would be nice to study strain diversity for these regimens by testing the drug regimens against other (clinical) M. tuberculosis strains. Finally, we previously only conducted studies of interactions and synergy mechanisms in two-drug combinations in the regimen. For instance, we previously found that TBI-166 and BDQ may disrupt the synthesis of ATP to kill bacteria quickly by dual actions on the respiratory chain of M. tuberculosis (28). However, due to the complexity of the three-drug combinations, we have not been able to elucidate how the three-drug combinations in this study exert a synergistic bactericidal effect, and further research is needed.
In conclusion, in this study, we found that OTB-658 can replace LZD in the BPaL or TBI-166 + BDQ + LZD regimen. The BDQ + PMD + OTB-658(50) and TBI-166 + BDQ + OTB-658(100) regimens have similar or slightly better short-term and long-term efficacy compared with BPaL, and further clinical trial studies should be performed.
MATERIALS AND METHODS
All experiments were performed at the Beijing Tuberculosis and Thoracic Tumor Research Institute (Beijing, China). Animal experiments were approved by the Animal Ethics Committee of the Beijing Chest Hospital-Affiliate of Capital Medical University, and all animal procedures were performed according to the Animal Care Guidelines of the Institutional Animal Care and Use Committee of Capital Medical University (Beijing, China).
Antimicrobial agents.
BDQ and PMD were purchased from Biochempartner (Shanghai, China). LZD was purchased from Sigma-Aldrich. TBI-166 and OTB-658 were provided by the Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences (Beijing, China).
Mycobacterial strain.
M. tuberculosis H37Rv was cultured in Middlebrook 7H9 broth (Difco, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) with 0.05% Tween 80 (Sigma-Aldrich) and 10% (vol/vol) oleic acid-albumin-dextrose-catalase (OADC; Becton, Dickinson and Company). After 2 weeks of incubation at 37°C and 5% CO2, the bacterial culture was used for further experiments.
Establishment of infection in mice.
All experiments used mice aged 6 to 8 weeks and weighing 18 g to 20 g, purchased from Beijing Vital River Laboratory Animal Technology Company (Beijing, China). Five mice were assigned to each cage and were housed at a temperature of 21 ± 2°C, humidity of 55% ± 15%, and under a 12 h/12 h light/dark cycle with easy access to food and filtered water. The mice were allowed to adapt to the environment for 5 days before the start of the experiment. All mice were aerosol-infected with mouse-passaged M. tuberculosis H37Rv by using an inhalation exposure system (099C A4224, Glas-Col, Terre Haute, IN, USA).
For experiment 1, 56 female C3HeB/FeJ mice were used. Three untreated mice were sacrificed 14 and 21 days after the infection to determine the baseline counts of bacteria implanted in lungs and at the start of treatment, respectively. Five mice from each group were sacrificed after 8 weeks of treatment to assess the bactericidal activity of each regimen. In addition, five mice were used to evaluate relapse after 12 weeks following the discontinuation of treatment at 8 weeks.
For experiment 2, 130 female BALB/c mice were used. Five untreated mice were sacrificed 3 and 14 days after infection to determine the baseline counts of bacteria implanted in lungs and at the start of treatment, respectively. Fifteen mice treated for 4 and 8 weeks in each of the three combined regimens were evaluated for relapse 12 weeks after drug withdrawal.
For experiment 3, 64 female BALB/c mice were used. Four untreated mice from each infection run were sacrificed at 10 days after infection to determine the bacterial counts at the start of treatment. Five mice from each group were sacrificed after 4 and 8 weeks of treatment to assess the bactericidal activity of each regimen.
Chemotherapy regimens.
In experiment 1, after 21 days of infection, C3HeB/FeJ mice were randomized into six groups (Table 3). In experiment 2, after 14 days of infection, BALB/c mice were randomized into four groups (Table 4). In experiment 3, after 10 days of infection, BALB/c mice were randomized into six groups (Table 5). All drugs were prepared in 0.5% sodium carboxymethyl cellulose (CMC) in distilled water. Drugs were administered at the following doses: BDQ, 25 mg/kg; PMD, 100 mg/kg; LZD, 100 mg/kg; TBI-166, 20 mg/kg; and OTB-658, 50 or 100 mg/kg. The drug dosages were obtained from previous experiments (12, 13).
TABLE 3
| Groupb | No. of mice sacrificed in each groupa | Total no. of mice | ||
|---|---|---|---|---|
| D-7 | D0 | W8 | ||
| Untreated | 3 | 3 | 5 | 11 |
| BpaL(100) | 5 | 5 | ||
| BDQ + PMD + OTB-658(50) | 5 + 5 | 10 | ||
| TBI-166 + BDQ + LZD(100) | 5 + 5 | 10 | ||
| TBI-166 + BDQ + OTB-658(50) | 5 + 5 | 10 | ||
| TBI-166 + BDQ + OTB-658(100) | 5 + 5 | 10 | ||
| Total no. of mice | 3 | 3 | 50 | 56 |
a
Time points: D-7 = 14 days after aerosol infection (n = 3), D0 = day of treatment, 21 days after infection (n = 3); W8 = treated for 8 weeks. D, day; W, week.
b
Drugs (abbreviations) and doses: bedaquiline (BDQ), 25 mg/kg; pretomanid (PMD), 100 mg/kg; linezolid (LZD), 100 mg/kg; TBI-166, 20 mg/kg; OTB-658(50), 50 mg/kg; OTB-658(100), 100 mg/kg; BpaL(100), BDQ + PMD + LZD(100).
TABLE 4
| Groupb | No. of mice sacrificed in each groupa | Total no. of mice | |||
|---|---|---|---|---|---|
| D-11 | D0 | W4 | W8 | ||
| Untreated | 5 | 5 | 10 | ||
| BpaL(100) | 5 + 15 | 5 + 15 | 40 | ||
| TBI-166 + BDQ + LZD(100) | 5 + 15 | 5 + 15 | 40 | ||
| TBI-166 + BDQ + OTB-658(100) | 5 + 15 | 5 + 15 | 40 | ||
| Total no. of mice | 5 | 5 | 60 | 60 | 130 |
a
Time points: D-11 = 3 days after aerosol infection (n = 5), D0 = day of treatment, 14 days after infection (n = 5); W4 = treated for 4 weeks; W8 = treated for 8 weeks. D, day; W, week.
b
Drugs (abbreviations) and doses: bedaquiline (BDQ), 25 mg/kg; pretomanid (PMD), 100 mg/kg; linezolid (LZD), 100 mg/kg; TBI-166, 20 mg/kg; OTB-658, 100 mg/kg; BpaL(100), BDQ + PMD + LZD(100).
TABLE 5
| Groupb | No. of mice sacrificed in each groupa | Total no. of mice | ||
|---|---|---|---|---|
| D0 | W4 | W8 | ||
| Untreated | 4 | 5 | 5 | 14 |
| TBI-166 | 5 | 5 | 10 | |
| LZD | 5 | 5 | 10 | |
| OTB-658 | 5 | 5 | 10 | |
| TBI-166 + LZD | 5 | 5 | 10 | |
| TBI-166 + OTB-658 | 5 | 5 | 10 | |
| Total no. of mice | 4 | 30 | 30 | 64 |
a
Time points: D0 = day of treatment, 10 days after infection (n = 4); W4 = treated for 4 weeks; W8 = treated for 8 weeks. D, day; W, week.
b
Drugs (abbreviations) and doses: linezolid (LZD), 100 mg/kg; TBI-166, 20 mg/kg; OTB-658, 100 mg/kg.
All drugs were administered orally by gavage five times per week (Monday to Friday) and for the three combined regimens, a single dose was 0.3 mL of CMC containing antibiotics. The negative-control groups (untreated) only received 0.2 mL of CMC containing no antibiotics, the monotherapy groups (TBI-166, LZD(100), OTB-658(100)), and two drug combination groups (TBI-166 + LZD(100), TBI-166 + OTB-658(100)) received 0.2 mL of CMC containing antibiotics, respectively.
Assessment of treatment effect.
The treatment effect was assessed based on the lungs and/or spleen CFU counts during treatments. Five mice from each group were sacrificed after 4 and/or 8 weeks treatment. The lungs or spleen were dissected and homogenized in 3.0 mL of sterile saline. Tissue homogenates were diluted 10-, 100-, and 1,000-fold, and 0.1 mL of undiluted homogenate or dilution was plated on 7H10 selective agar plates enriched with 10% OADC enrichment medium (Difco) and supplemented with ampicillin (50 μg/mL), polymyxin B (33.3 μg/mL), trimethoprim (20 μg/mL), and cycloheximide (100 μg/mL) to prevent contamination by other bacteria. To limit the consequences of TBI-166 and BDQ carryover, lung homogenates from the mice treated with BDQ and TBI-166 were plated on 7H10 selective agar supplemented with 0.4% (wt/vol) activated charcoal, which adsorbed the residual TBI-166 and BDQ. All plates were incubated at 37°C with 5% CO2 for 4 weeks before the CFU counts. The same plating scheme was also used for the relapse assessment.
Statistical analysis.
CFU counts (x) were log transformed as log10 (x + 1) before analysis. Comparisons of CFU means between different groups were analyzed by one-way analysis of variance with Dunnett’s post hoc test to correct for multiple comparisons. The relapse rates were compared using Fisher’s exact test. The significance level was 0.05. SPSS (20.0, version for Windows, IBM, Armonk, NY, USA) was used for all statistical analyses.
ACKNOWLEDGMENTS
We thank the Institute of Materia Medica, Peking Union Medical College & Chinese Academy of Medical Sciences for providing TBI-166 and OTB-658.
This work was supported by the National Natural Science Foundation of China (82173862) and Beijing Hospitals Authority Clinical Medicine Development of Special Funding Support (ZYLX202123).
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Published In
Antimicrobial Agents and Chemotherapy
Volume 67 • Number 2 • 16 February 2023
eLocator: e01399-22
PubMed: 36622240
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© 2023 American Society for Microbiology. All Rights Reserved.
History
Received: 15 October 2022
Returned for modification: 10 November 2022
Accepted: 18 December 2022
Published online: 9 January 2023
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2023.
Efficacy of Replacing Linezolid with OTB-658 in Anti-Tuberculosis Regimens in Murine Models. Antimicrob Agents Chemother
67:e01399-22.
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