ABSTRACT
TBI-166, derived from riminophenazine analogues, shows more potent anti-TB activity than clofazimine and is being assessed against tuberculosis (TB) in a phase IIa clinical trial in China. Preclinical regimen studies containing TBI-166 will support the phase IIb clinical trials of TBI-166. In the present study, we compared the efficacy in three murine TB models of an all-oral drug-resistant TB drug regimen of TBI-166 with bedaquiline (BDQ) and pyrazinamide (PZA) with the first-line regimen of isoniazid (INH) with rifampin (RFP) and PZA (HRZ regimen), the most effective reported TBI-166-containing regimen of TBI-166 with BDQ and linezolid (LZD), and the Nix-TB clinical trial regimen of BDQ with pretomanid and LZD (BPaL regimen). In the C3HeB/FeJ murine TB model, for the TBI-166+BDQ+PZA regimen, the lungs of mice were culture negative at 4 weeks, and there were no relapses at 8 weeks of treatment. The reduction in bacterial burden and relapse rate were greater than those of the HRZ regimen and the TBI-166+BDQ+LZD regimen. Compared with the BPaL regimen, the TBI-166+BDQ+PZA regimen had similar or stronger early bactericidal activity, bactericidal activity, and sterilizing activity in the BALB/c murine TB model. The bacterial burden in the TBI-166+BDQ+PZA regimen group decreased significantly more than that in the BPaL regimen group and was almost or totally relapse free (<13.33% after 8 weeks). In conclusion, oral short-course three-drug regimens, including TBI-166 with high efficacy, were identified. The TBI-166+BDQ+PZA regimen is recommended for further study in a TBI-166 phase IIb clinical trial.
KEYWORDS: TBI-166, tuberculosis, murine model, BALB/c mice, C3HeB/FeJ mice, regimen
INTRODUCTION
It is estimated that in 2020, 1.5 million people died of tuberculosis (TB), and more than 10 million people suffered from the disease worldwide, among which there were nearly 160,000 drug-resistant tuberculosis (DR-TB) cases. The treatment of DR-TB requires a combination of more than three drugs. The greatest challenge for clinical practitioners is that among DR-TB cases, 54% are multidrug-resistant tuberculosis (MDR-TB), and 30% are extensively drug-resistant tuberculosis (XDR-TB) (1). Because of the prolonged treatment duration, low success rate, and side effects of anti-TB drugs and consequent poor patient adherence, the DR-TB epidemic has become an enormous problem. Therefore, shorter, less toxic, and more effective regimens are urgently needed.
TBI-166 is a new drug candidate derived from the optimization of riminophenazine analogs (2). It has higher bactericidal activity and causes fewer adverse drug reactions than the lead riminophenazine compound, clofazimine (CLO) (3, 4). Our group had already reported a high efficacy for TBI-166 monotherapy and its combination treatment regimen both in vitro and in vivo. The in vivo lung CFU reduction in mice showed that in combination with TBI-166, the bactericidal activities of bedaquiline (BDQ) or pyrazinamide (PZA) were significantly increased (5). Furthermore, five TBI-166-containing regimens composed of at least three drugs had significantly higher bactericidal activity than that of the standard first-line isoniazid (INH) with rifampin (RFP) and PZA (HRZ) regimen, and the most effective combination was the TBI-166 with BDQ and linezolid (LZD) regimen (5). But the combination treatment regimens containing TBI-166 evaluated were limited in the same study.
Based on these results, the potential efficacy of a drug regimen consisting of TBI-166, BDQ, and PZA should be investigated. In the current study, three independent, long-term, relapse-based chemotherapy studies were conducted in murine TB models, in which the BDQ with pretomanid (PMD) and LZD (BPaL) regimen was used as the positive control.
RESULTS
Experiment 1: comparison of TBI-166+BDQ+LZD and TBI-166+BDQ+PZA regimens with first-line HRZ regimen in C3HeB/FeJ mice.
Based on the synergistic effects reported in combinations of TBI-166+BDQ, TBI-166+PZA, and BDQ+PZA, and to minimize the possible adverse drug reactions related to LZD (6), the efficacy of the TBI-166+BDQ+PZA regimen was compared with those of the first-line HRZ regimen and the TBI-166+BDQ+LZD regimen in a C3HeB/FeJ murine TB model. The lung tissues of C3HeB/FeJ mice can produce hypoxic, well-defined granulomas, and regimens are expected to demonstrate activity in the presence of granulomas.
The CFU counts for the lungs and spleen in the C3HeB/FeJ mice infected with M. tuberculosis H37Rv at 4 and 8 weeks after different treatments are presented in Table 1. Treatment began 6 weeks after infection (D0) when the mean CFU count was 5.63 log10 CFU in the lungs and 4.61 log10 CFU in the spleen. The mean bacterial burden of the untreated mice remained high throughout the trial, whereas after 4 weeks of treatment, the mean bacterial burden in the first-line HRZ regimen treated mice decreased to 2.02 and 2.21 log10 CFU in the lungs and spleen, respectively. All lungs were culture negative, and 80% of the spleens were culture negative in both the TBI-166+BDQ+LZD and TBI-166+BDQ+PZA groups after 4 weeks of treatment; the mean CFU count was 1.48 log10 CFU in the spleen. All three treatment groups were culture negative after 8 weeks (Table 1).
TABLE 1.
Lung and spleen CFU counts at the indicated time points in C3HeB/FeJ mice
| Groupb | Mean log10 CFU count (positive lung or spleen culture) at:a |
||
|---|---|---|---|
| D0 | W4 | W8 | |
| Lung | |||
| Untreated | 5.63 ± 0.36 (5/5) | 4.93 ± 0.42 (5/5) | |
| HRZ | 2.02 ± 0.41 (5/5) | Negative | |
| TBI-166+BDQ+LZD | Negative | Negative | |
| TBI-166+BDQ+PZA | Negative | Negative | |
| Spleen | |||
| Untreated | 4.61 ± 0.20 (5/5) | 4.35 ± 0.13 (5/5) | |
| HRZ | 2.21 ± 0.17 (5/5) | Negative | |
| TBI-166+BDQ+LZD | 1.48 (1/5) | Negative | |
| TBI-166+BDQ+PZA | 1.48 (1/5) | Negative | |
Time points are shown as days (D0) or weeks (W4 or W8) of treatment. The start of the treatment (D0) began 6 weeks after infected with M. tuberculosis H37Rv. Values are means ± standard deviations with proportion of positive cultures shown in parentheses; n = 5.
Drugs (abbreviations) and doses are as follows: isoniazid (INH), 10 mg/kg; rifampin (RFP), 10 mg/kg; pyrazinamide (PZA), 150 mg/kg; TBI-166, 20 mg/kg; bedaquiline (BDQ), 25 mg/kg; linezolid (LZD), 100 mg/kg; HRZ, INH+RFP+PZA.
Five mice each were taken from TBI-166+BDQ+LZD and TBI-166+BDQ+PZA groups, and the treatment was discontinued for 12 weeks after 8 weeks of treatment. Lung and spleen homogenates from the mice were cultured on 7H10 plates containing activated carbon. Three of the five mice in the TBI-166+PMD+LZD group relapsed, whereas there were no culture-positive relapses in the TBI-166+BDQ+PZA group.
Experiment 2: assessment of the sterilizing activity of the TBI-166+BDQ+PZA regimen.
To explain the higher efficacy of the TBI-166+BDQ+PZA regimen over the TBI-166+BDQ+LZD regimen observed in experiment 1, in mice infected with M. tuberculosis H37Rv, the bacterial burden and sterilizing activity of the TBI-166+BDQ+PZA regimen were observed in a BALB/c murine TB model over 8 weeks.
The bacterial burden measurements showed that the TBI-166+BDQ+PZA and BPaL groups were culture negative for the lungs and spleen after 4 and 8 weeks of treatment. In the sterilizing activity measurements, relapse occurrence was determined after 12 weeks following the discontinuation of a 4- or 8-week treatment regimen with TBI-166+BDQ+PZA or BPaL. Both regimens showed high bactericidal activities, with culture negativity observed in the lungs and spleen after 4 and 8 weeks treatment. The relapse rate was significantly lower for the TBI-166+BDQ+PZA regimen (21.43%) than for the BPaL regimen (69.23%) for 4 weeks of treatment (P < 0.001), and all mice were completely cured with no relapse for both regimens after 8 weeks of treatment (Table 2).
TABLE 2.
Relapse of BALB/c mice treated with the BPaL or TBI-166+BDQ+PZA regimen
| Groupb | Mean log10 CFU count at: |
Proportion of mice that relapsed after treatment (% relapse in group) at:a |
|||
|---|---|---|---|---|---|
| D0 | W4 | W8 | W4 (+W12) | W8 (+W12) | |
| Untreated | 3.62 ± 0.30 (5/5) | ||||
| BPaL | Negative | Negative | 9/13 (69.23)c | 0/15 (0) | |
| TBI-166+BDQ+PZA | Negative | Negative | 3/14 (21.43)c,d | 0/15 (0) | |
Time points are shown as days (D0) or weeks (W4 or W8) of treatment. The start of the treatment (D0) began 4 weeks after infection with M. tuberculosis H37Rv. Mouse lung and spleen homogenates were cultured on 7H10 plates containing activated carbon at the end of 12 weeks following the discontinuation of the treatment regimen of 4 weeks (W4 + W12) or 8 weeks (W8 + W12). n = 15.
Drugs (abbreviations) and doses are as follows: pyrazinamide (PZA), 150 mg/kg; TBI-166, 20 mg/kg; bedaquiline (BDQ), 25 mg/kg; linezolid (LZD), 100 mg/kg; pretomanid (PMD), 100 mg/kg; BPaL, BDQ+PMD+LZD.
One mouse in the TBI-166+BDQ+PZA group and two mice in the BPaL group died due to gavage accidents.
P < 0.001.
Experiment 3: comparison of BPaL and TBI-166+BDQ+PZA in BALB/c mice.
To clarify the difference between the TBI-166+BDQ+PZA and BDQ+PMD+LZD regimens, the bacterial burdens 2, 4, and 8 weeks after treatment and the relapse rate 4 and 8 weeks after treatment were examined.
During the whole chemotherapy period, both regimens were well tolerated, as evaluated by the maintenance of body weight, and all mice showed no abnormalities in gross appearance. The bacterial burdens in the lungs and spleen after 2, 4, and 8 weeks of treatment are shown in Table 3. Three days after aerosol infection, the mean CFU count was 2.26 log10 CFU in the lungs, which increased to 4.28 log10 CFU at the start of treatment 4 weeks after infection. The mean CFU count remained high in the untreated group (negative control) throughout the trial. In mice infected with M. tuberculosis H37Rv, the treatment groups had significantly lower mean CFU counts than the negative-control group after 2, 4, and 8 weeks (P < 0.001) of treatment (Fig. 1). The mean bacterial burden in the positive-control group treated with BPaL decreased to 1.51 log10 CFU after 4 weeks of treatment and became culture negative at 8 weeks. Compared with the positive-control group, after 2 weeks of treatment, the TBI-166+BDQ+PZA group showed an additional reduction of approximately 1 log10 CFU in the lungs of BALB/c mice (P < 0.05). After 4 weeks of treatment, 80% (4/5) of mice in the TBI-166+BDQ+PZA group had become culture negative, and the CFU count in the lungs was 1.18 log10 CFU. In contrast, 40% (2/5) of mice in the BPaL group had become culture negative, and the CFU count in the lungs was approximately 1.51 log10 CFU. All treatment groups were culture negative at the end of 8 weeks of treatment.
TABLE 3.
CFU counts in the lungs and spleen at the indicated time points in BALB/c mice
| Groupb | Mean lung log10 CFU counts (positive lung or spleen culture) at:a |
||||
|---|---|---|---|---|---|
| D28 | D0 | W2 | W4 | W8 | |
| Lungs | |||||
| Untreated | 2.26 ± 0.07 (3/3) | 4.82 ± 0.13 (3/3) | 5.31 ± 0.25 (5/5) | 5.20 ± 0.47 (5/5) | 4.32 ± 0.50 (5/5) |
| BPaL | 3.99 ± 0.47 (5/5) | 1.51 ± 0.35 (3/5) | Negative | ||
| TBI-166+BDQ+PZA | 3.20 ± 0.35 (5/5) | 1.18 (1/5) | Negative | ||
| Spleen | |||||
| Untreated | Negative | 2.38 ± 0.16 (3/3) | 3.13 ± 0.29 (5/5) | 3.02 ± 0.36 (5/5) | 2.68 ± 0.24 (5/5) |
| BPaL | 1.64 ± 0.29 (4/5) | Negative | Negative | ||
| TBI-166+BDQ+PZA | 2.24 ± 0.27 (5/5) | Negative | Negative | ||
Time points are shown as day (D28 or D0) or week (W2, W4, or W8) of treatment. The start of the treatment (D0) began 4 weeks after infected with M. tuberculosis H37Rv. Values are means ± standard deviation with proportion of positive cultures shown in parentheses; n = 5.
Drugs (abbreviations) and doses are as follows: pyrazinamide (PZA), 150 mg/kg; TBI-166, 20 mg/kg; bedaquiline (BDQ), 25 mg/kg; linezolid (LZD), 100 mg/kg; BPaL, BDQ+PMD+LZD.
FIG 1.
Changes in lung CFU counts in BALB/c mice infected with M. tuberculosis H37Rv treated with BPaL or TBI-166-containing regimens. Pyrazinamide, PZA; bedaquiline, BDQ; pretomanid, PMD; linezolid, LZD; BPaL, BDQ+PMD+LZD. Values are means ± standard deviation; n = 5. W, week. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Relapse was also assessed from the lungs and spleen 12 weeks after completion of 4 and 8 weeks of treatment in BALB/c mice (Table 4). The relapse rate after 4 weeks of treatment was higher in the BPaL group (13/15 [86.67%]) than in the TBI-166+BDQ+PZA group (11/15 [73.34%]), although the difference was not statistically significant (P = 0.361). When the treatment duration was extended to 8 weeks, the relapse rate was lower in the BPaL group (1/15 [6.67%]) than the TBI-166+BDQ+PZA group (2/15 [13.34%]), although the difference was not significant (P = 1.0) (Table 4). The TBI-166+BDQ+PZA regimen presented the same trends in early bactericidal activity (EBA), bactericidal activity, and sterilizing activity.
TABLE 4.
Relapse rate of BALB/c mice in lungs and spleen treated with BPaL or TBI-166+BDQ+PZA for 4 and 8 weeks
| Groupb | Relapse rate after treatment (% relapse in group) at:a |
|
|---|---|---|
| W4 (+W12) | W8 (+W12) | |
| BPaL | 13/15 (86.67) | 1/15 (6.67) |
| TBI-166+BDQ+PZA | 11/15 (73.34) | 2/15 (13.33) |
Time points are shown as day (D0) or week (W4 or W8) of treatment. The start of the treatment (D0) began 4 weeks after infection with M. tuberculosis H37Rv. Lung and spleen homogenates were cultured on 7H10 plates containing activated carbon at the end of 12 weeks of the discontinuance of the regimen treatment of 4 (W4 + W12) or 8 (W8 + W12) weeks.
Drugs (abbreviations) and doses are as follows: pyrazinamide (PZA), 150 mg/kg; TBI-166, 20 mg/kg; bedaquiline (BDQ), 25 mg/kg; linezolid (LZD), 100 mg/kg; pretomanid (PMD), 100 mg/kg; BPaL, BDQ+PMD+LZD.
DISCUSSION
Worldwide TB epidemics remain severe, and the emergence of MDR-TB and XDR-TB presents a major challenge for efforts to eradicate TB; thus, there is an urgent need for safe, effective drugs and regimens for treating TB. TBI-166 is a drug candidate obtained by optimizing the riminophenazine CLO, and it causes less skin discoloration than CLO despite its higher tissue accumulation (3, 4). In our previous studies, TBI-166 showed higher bactericidal activity than CLO both in vitro and in vivo and presented great bactericidal activity in the CLO-resistant M. tuberculosis with Rv0678 gene mutation (3), and TBI-166 is in phase IIa clinical trials owing to its better efficiency and safety than CLO. Preclinical studies on regimens containing TBI-166 have achieved important progress in preparation for the phase IIb trials. Our previous study clarified that of five TBI-166-containing regimens, the TBI-166+BDQ+LZD regimen was the best regimen containing BDQ and TBI-166. This regimen showed stronger bactericidal and sterilizing activity in both BALB/c and C3HeB/FeJ murine TB models than the standard first-line HRZ regimen (5). To develop and evaluate regimens containing TBI-166 further, in the present study, we systematically assessed the bactericidal and sterilizing activity of the TBI-166+BDQ+PZA regimen in murine TB models.
To examine the bactericidal and sterilizing activity of the TBI-166+BDQ+PZA regimen, we used the C3HeB/FeJ murine TB model to better mimic the pathophysiological conditions found in caseating human lung lesions. The lung tissues of C3HeB/FeJ mice can produce hypoxic, well-defined granulomas and exhibit caseous necrosis, and the microenvironmental conditions in granulomas may decrease drug efficacy, which may not be reflected in more conventional BALB/c murine TB models (7). The present study also confirmed that the TBI-166+BDQ+LZD regimen has stronger activity in reducing bacterial burden than the standard HRZ regimen. Regarding sterilizing activity, when the HRZ group still had a 40% relapse rate at 12 weeks of discontinuation after 8 weeks of treatment, the TBI-166+BDQ+LZD group had no relapse after 8 weeks of treatment (5). Compared with the strongest regimen of TBI-166+BDQ+LZD identified in the previous study (5) and the first-line HRZ regimen, we concluded that the bactericidal and sterilizing activities of the TBI-166+BDQ+PZA regimen were stronger. The lungs in the TBI-166+BDQ+PZA and TBI-166+BDQ+LZD groups were all culture negative after 4 weeks, indicating that the bactericidal activity of the TBI-166+BDQ+PZA regimen was similar to or stronger than that of the TBI-166+BDQ+LZD regimen. Furthermore, there were no relapses in the TBI-166+BDQ+PZA group after 8 weeks of treatment, whereas the relapse rate was 60% in the TBI-166+BDQ+LZD group, indicating that the TBI-166+BDQ+PZA regimen had stronger sterilizing activity than the TBI-166+BDQ+LZD regimen.
To examine the efficacy of the TBI-166+BDQ+PZA regimen further, the BPaL regimen was used as the positive control in experiments 2 and 3. We assessed the bactericidal and sterilizing activities against TB in two BALB/c mouse models. These results reinforced our findings from experiment 1 that the TBI-166+BDQ+PZA regimen had high bactericidal and sterilizing activity and is promising as an oral short-course (e.g., 6 month) regimen for DR-TB.
Based on experiment 1 and other murine studies (8, 9), BALB/c mice were expected to be nearly culture negative after 4 weeks of treatment with the TBI-166+BDQ+PZA regimen and 8 weeks of treatment with the BPaL regimen. Therefore, we evaluated the bacterial burden of mice and sterilizing activity for the TBI-166+BDQ+PZA regimen after 4 or 8 weeks of treatment compared with the BPaL regimen.
The results of experiments 1 and 2 indicated that the TBI-166+BDQ+PZA regimen were nearly or totally culture negative after 4 weeks treatment, suggesting that we could focus on the early bactericidal activity (EBA) of these regimens. In our previous research (5) and similar to other murine model studies (10), in the early phase of treatment, the efficacy appeared to be driven mainly by BDQ, and LZD showed only minor or no bactericidal activity. The combination of PZA with TBI-166 and BDQ increased the EBA of the regimen, and the EBA of the TBI-166+BDQ+PZA regimen was statistically higher than that of the BPaL regimen. At the same time, the TBI-166+BDQ+PZA regimen had good activity against M. tuberculosis H37Rv in reducing the bacterial burden at each time point of the full-course treatment at 2 weeks, 4 weeks, and 8 weeks.
The BPaL regimen had superior bactericidal and sterilizing activity over the first-line INH+RFP+PZA with ethambutol (HRZE) regimen (11), and the TBI-166+BDQ+PZA regimen had similar or stronger sterilizing activity compared with the BPaL regimen in the murine TB model. After 4 weeks of treatment, the TBI-166+BDQ+PZA group had a lower relapse rate than the BPaL group, and both regimens were culture negative or nearly relapse free after 8 weeks of treatment. The BPaL regimen, used in the Nix-TB clinical trial, has the potential to transform the treatment of DR-TB, and it shows promise as an oral short-course (e.g., 6 month) regimen for MDR-TB and XDR-TB. The TBI-166+BDQ+PZA regimen showed similar or stronger EBA, bactericidal, and sterilizing activity than the BPaL regimen. The bactericidal and sterilizing activity of the TBI-166+BDQ+PZA regimen should accelerate sputum culture conversion and reduce the relapse rate of TB, and this regimen could be converted into an all-oral three-drug short-course regimen lasting 6 months for MDR-TB and XDR-TB.
The superior efficacy of the TBI-166+BDQ+PZA regimen has several possible explanations. First, BDQ, TBI-166, and PZA showed intense anti-TB activity in our study and previous studies. The congener of TBI-166, CLO, also increased the activity of BDQ+PZA significantly (12). Second, synergistic effects may arise from BDQ, PZA, and TBI-166 inhibiting key pathways in the oxidative phosphorylation and ATP synthesis of Mycobacterium tuberculosis, such as PZA binding to aspartate decarboxylase (13) and BDQ inhibiting the proton pump of mycobacterial ATP synthase subunit c. Third, TBI-166 may be a CYP3A4 inhibitor, similar to CLO (14). Because BDQ is a substrate of CYP3A4, the drug-drug interaction between TBI-166 and BDQ may increase blood BDQ exposure and decrease the amount of the major toxic metabolism product N-monodesmethyl bedaquiline (M2), increasing the efficacy and reducing the adverse drug reaction of BDQ. Fourth, we previously found that TBI-166 combined with BDQ decreased ATP content in bacteria significantly and increased the bacterial reactive oxygen species (ROS) content significantly compared with BDQ or TBI-166 alone. Thus, the synergistic bactericidal mechanism of TBI-166 and BDQ could be related to the increased accumulation of ROS and the inhibition of ATP synthesis. Fifth, the accumulation of BDQ and TBI-166 in tissues is high, their half-life in vivo is long, and, thus, the redistribution of accumulated BDQ and TBI-166 can cause a bactericidal effect. This would also partly explain why BDQ- and TBI-166-containing combinations have high sterilizing activity against intracellular bacteria.
Our present findings and the results of previous work support the idea that because of the synergistic activity of the combination of BDQ and PZA, it will form the foundation of many regimens against DR-TB (15, 16). The bactericidal activity of the combination of BDQ and PZA is superior to that of the standard first-line regimen (17). PZA has been used as the “partner” for BDQ in multiple regimens, such as BPaMZ (BDQ, PMD, moxifloxacin, and PZA), which also has superior efficacy to the standard first-line regimen (18). The synergistic effect of adding TBI-166 to this partner substantially increases the anti-TB activity of the combination of BDQ and PZA.
It is important to balance the EBA, sterilizing activity, safety, and simple administration of a drug combination. It usually takes 9 to 24 months with regimens containing at least four drugs, including one injectable agent, to treat patients with MDR-TB (19). In comparison, the fully oral short-course, three-drug TBI-166+BDQ+PZA regimen may be an effective and simpler choice for MDR-TB patients (20). We recommend the use of TBI-166-containing regimens at the beginning of treatment to minimize the development of drug resistance.
However, there are several limitations to the current study. First, the bacterial load in the mice models was relatively low. In each BALB/c mouse model, the mean lung burden at the beginning of treatment was 4.82 log10 CFU at 4 weeks and 5.63 log10 CFU at 6 weeks following aerosol infection. Thus, all mice in the positive-control and TBI-166-containing regimens were approaching culture-negative status after 4 weeks of treatment. These results suggested that the anti-TB activity of the regimen was strong, but it was difficult to distinguish which was the most effective after 4 and 8 weeks. We think that a higher inoculum might extend the duration of therapy; however, the conclusion that the TBI-166+BDQ+PZA regimen is the most effective regimen in 3 experiments will not change. Another limitation was the use of the drug-sensitive M. tuberculosis H37Rv strain to assess the DR-TB regimen. However, many previous studies of DR-TB treatment regimens being evaluated in clinical trials have found the same trends in both drug-resistant and drug-sensitive strains (21). Some of these regimens are already showing good translation to clinical practice. Also, the lack of pharmacokinetic data is a limitation; pharmacokinetic data would have assisted interpretation of the potential pharmacokinetic and pharmacodynamic drug-drug interactions (DDIs) of these results for the regimen. PK research of the three-drug regimen is the subject of our future research.
In conclusion, the TBI-166+BDQ+PZA regimen has strong bactericidal and sterilizing activity and is a promising combination for the treatment of DR-TB. The strong bactericidal and sterilizing activity of the TBI-166+BDQ+PZA regimen in murine TB models suggests that in the clinic, the sputum culture conversion rate will be high and the TB recurrence rate will be low; thus, it is worth evaluating TBI-166 in phase IIb clinical trials as an oral short course (e.g., 6 months) in a three-drug regimen for MDR-TB and XDR-TB.
MATERIALS AND METHODS
All experiments were performed at the Beijing Tuberculosis and Thoracic Tumor Research Institute (Beijing, China). Animal experiments in this study 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.
INH, RFP, BDQ, PMD, LZD, and PZA were purchased from Sigma-Aldrich. TBI-166 was provided by the Institute of Materia Medica, Peking Union Medical College, and Chinese Academy of Medical Sciences (Beijing, China).
Mycobacterial strain.
M. tuberculosis H37Rv (ATCC 27294) was grown in 7H9 broth supplemented with 10% Middlebrook acid-albumin-dextrose-catalase (OADC) enrichment medium (Difco), 0.2% glycerol, and 0.05% Tween 80. Log-phase cultures incubated at 37°C with 5% CO2 were used in all studies.
Establishment of infection in mice.
For experiment 1, female C3HeB/FeJ mice, aged 6 weeks and weighing 18 to 20 g, were purchased from Beijing Vital River Laboratory Animal Technology Company. The mice were assigned to cages at five mice per cage and allowed to acclimatize to the environment for 1 week before the experiment. All 51 female C3HeB/FeJ mice were aerosol infected with mouse-passaged M. tuberculosis H37Rv by using an inhalation exposure system (099C A4224; Glas-Col). Three untreated mice were sacrificed at both 10 days and 6 weeks after the infection to determine the baseline counts of bacteria implanted in lungs and at the start of treatment, respectively. Based on prior experience (5), tuberculosis mice were expected to be culture negative or nearly relapse free in groups containing TBI-166+BDQ after 8 weeks of treatment. Five mice from each group were sacrificed after 4 or 8 weeks of treatment to assess the bactericidal activity of each regimen. In addition, five mice treated for 8 weeks in each of the three combined regimens were evaluated for relapse 12 weeks after drug withdrawal. Bacterial load was assessed after completing 4 and 8 weeks of treatment in C3HeB/FeJ mice to evaluate bactericidal activities and relapse rate at after 12 weeks drug withdrawal after 8 weeks treatment to evaluate sterilizing activity.
For experiment 2, 86 female BALB/c mice, aged 6 weeks and weighing 18 to 20 g, were purchased from Beijing Vital River Laboratory Animal Technology Company. The mice were assigned to cages at five mice per cage and allowed to acclimatize to the environment for 1 week before the experiment. The housing was kept at a constant temperature and humidity using an air-conditioning system. Mice were aerosol infected with mouse-passaged M. tuberculosis H37Rv by using an inhalation exposure system (099C A4224). Three untreated mice were sacrificed both at 10 days and 4 weeks after the infection to determine the baseline counts of bacteria implanted in lungs and at the start of treatment, respectively. Based on prior experience (5) and experiment 1, tuberculosis mice were expected to be culture negative or nearly relapse free in the TBI-166+BDQ+PZA regimen after 8 weeks of treatment. Five mice from each group were sacrificed after 4 or 8 weeks of treatment to assess the bactericidal activity of each regimen. Fifteen mice treated for 4 and 8 weeks in each of the two combined regimens were evaluated for relapse 12 weeks after drug withdrawal.
For experiment 3, female BALB/c mice, aged 6 weeks and weighing 18 to 20 g, were purchased from Beijing Vital River Laboratory Animal Technology Company. Mice were assigned to cages at five mice per cage and allowed to acclimatize to the environment for 1 week before the experiment. All 111 mice were aerosol infected with mouse-passaged M. tuberculosis H37Rv by using an inhalation exposure system (099C A4224). Three untreated mice from each infection run were sacrificed at both 3 days and 4 weeks after infection to determine the bacterial counts at the beginning of infection and at the start of treatment, respectively. Based on all prior experience, tuberculosis mice were expected to be culture negative and relapse free in the TBI-166+BDQ+PZA group after 8 weeks of treatment. Five mice from each group were sacrificed after 2 weeks of treatment to assess the EBA and 4 or 8 weeks of treatment to assess the bactericidal activity of each regimen. In addition, 15 mice treated for 4 and 8 weeks in each of the three combined regimens were evaluated for relapse 12 weeks after drug withdrawal. Relapse rate at 12 weeks drug withdrawal after 4 and 8 weeks treatment were evaluated sterilizing activity.
Chemotherapy regimens.
In experiment 1, after 6 weeks of infection, BALB/c mice were randomized into 4 groups (Table 5). In experiment 2, after 4 weeks of infection, BALB/c mice were randomized into 2 groups, consisting of 6 groups for BPaL and 6 groups for TBI-166+BDQ+PZA (Table 6). In experiment 3, after 4 weeks of infection, BALB/c mice were randomized into 3 groups (Table 7). All drugs except PZA were prepared in 0.4% sodium carboxymethyl cellulose (CMC) in distilled water, and PZA was prepared in distilled water as a single drug. 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 PZA, 150 mg/kg. The dosage of TBI-166 was obtained from the previous experiment (3, 5).
TABLE 5.
Experimental design used in experiment 1
| Groupb | No. of mice sacrificed at:a |
Total no. of mice | |||
|---|---|---|---|---|---|
| D42 | D0 | W4 | W8c | ||
| Untreated | 3 | 3 | 5 | 11 | |
| HRZ | 5 | 5 | 10 | ||
| TBI-166+BDQ+LZD | 5 | 5 + 5 | 15 | ||
| TBI-166+BDQ+PZA | 5 | 5 + 5 | 15 | ||
| Total no. of mice | 3 | 3 | 20 | 25 | 51 |
D, day; W, week.
Drugs (abbreviations) and doses are as follows: bedaquiline (BDQ), 25 mg/kg; pretomanid (PMD), 100 mg/kg; linezolid (LZD), 100 mg/kg; TBI-166, 20 mg/kg; pyrazinamide (PZA), 150 mg/kg; HRZ, INH+RFP+PZA.
Five mice were assessed the bactericidal activity and five mice were evaluated sterilizing activity.
TABLE 6.
Experimental design used in experiment 2
| Groupb | No. of mice sacrificed at:a |
Total no. of mice | |||
|---|---|---|---|---|---|
| D42 | D0 | W4c | W8c | ||
| Untreated | 3 | 3 | 6 | ||
| BPaL | 5 + 15 | 5 + 15 | 40 | ||
| TBI-166+BDQ+PZA | 5 + 15 | 5 + 15 | 40 | ||
| Total no. of mice | 3 | 3 | 40 | 40 | 86 |
D, day; W, week.
Drugs (abbreviations) and doses are as follows: bedaquiline (BDQ), 25 mg/kg; pretomanid (PMD), 100 mg/kg; linezolid (LZD), 100 mg/kg; TBI-166, 20 mg/kg; pyrazinamide (PZA), 150 mg/kg; BPaL, BDQ+PMD+LZD.
Five mice were assessed the bactericidal activity and fifteen mice were evaluated sterilizing activity.
TABLE 7.
Experimental design used in experiment 3
| Groupb | No. of mice sacrificed at:a |
Total no. of mice | ||||
|---|---|---|---|---|---|---|
| D28 | D0 | W2 | W4c | W8c | ||
| Untreated | 3 | 3 | 5 | 5 | 5 | 21 |
| BPaL | 5 | 5 + 15 | 5 + 15 | 45 | ||
| TBI-166+BDQ+PZA | 5 | 5 + 15 | 5 + 15 | 45 | ||
| Total no. of mice | 3 | 3 | 15 | 45 | 45 | 111 |
D, day; W, week.
Drugs (abbreviations) and doses are as follows: bedaquiline (BDQ), 25 mg/kg; pretomanid (PMD), 100 mg/kg; linezolid (LZD), 100 mg/kg; TBI-166, 20 mg/kg; pyrazinamide (PZA), 150 mg/kg; BPaL, BDQ+PMD+LZD.
Five mice were assessed the bactericidal activity and fifteen mice were evaluated sterilizing activity.
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. Negative-control groups received 0.2 mL of CMC. Positive-control groups received BPaL in 0.3 mL CMC.
Assessment of treatment effect.
The treatment effect was assessed based on the lung and spleen CFU counts during treatments. Five mice from each group were sacrificed after 2, 4, and 8 weeks treatment. After the mice were killed, the lungs or spleen were dissected and homogenized in 3.0 mL of sterilizing saline. Tissue homogenates were diluted 10-, 100-, and 1,000-fold, and 0.1 mL of undiluted homogenate or dilution was plated on selective 7H10 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 (200 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 absorbed the residual TBI-166. 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 recurrence 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 (version 19.0 for Windows; SPSS) was used for all statistical analyses.
ACKNOWLEDGMENTS
We thank the Institute of Materia Medica, Peking Union Medical College, and Chinese Academy of Medical Sciences for providing TBI-166.
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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