Bedaquiline and Pyrazinamide Treatment Responses Are Affected by Pulmonary Lesion Heterogeneity in Mycobacterium tuberculosis Infected C3HeB/FeJ Mice

Abstract

BALB/c and Swiss mice are routinely used to validate the effectiveness of tuberculosis drug regimens, although these mouse strains fail to develop human-like pulmonary granulomas exhibiting caseous necrosis. Microenvironmental conditions within human granulomas may negatively impact drug efficacy, and this may not be reflected in non-necrotizing lesions found within conventional mouse models. The C3HeB/FeJ mouse model has been increasingly utilized as it develops hypoxic, caseous necrotic granulomas which may more closely mimic the pathophysiological conditions found within human pulmonary granulomas. Here, we examined the treatment response of BALB/c and C3HeB/FeJ mice to bedaquiline (BDQ) and pyrazinamide (PZA) administered singly and in combination. BALB/c mice consistently displayed a highly uniform treatment response to both drugs, while C3HeB/FeJ mice displayed a bimodal response composed of responsive and less-responsive mice. Plasma pharmacokinetic analysis of dissected lesions from BALB/c and C3HeB/FeJ mice revealed that PZA penetrated lesion types from both mouse strains with similar efficiency. However, the pH of the necrotic caseum of C3HeB/FeJ granulomas was determined to be 7.5, which is in the range where PZA is essentially ineffective under standard laboratory in vitro growth conditions. BDQ preferentially accumulated within the highly cellular regions in the lungs of both mouse strains, although it was present at reduced but still biologically relevant concentrations within the central caseum when dosed at 25 mg/kg. The differential treatment response which resulted from the heterogeneous pulmonary pathology in the C3HeB/FeJ mouse model revealed several factors which may impact treatment efficacy, and could be further evaluated in clinical trials.

Keywords: C3HeB/FeJ; bedaquiline; mouse models; pyrazinamide; tuberculosis.

Conflict of interest statement

The authors declare the following competing financial interest(s): K.A. is an employee of Janssen Pharmaceutica, the company that developed bedaquiline.

Figures

Figure 1

PZA activity in BALB/c and C3HeB/FeJ mice. Plots represent log10 CFU determinations from the lungs of individual BALB/c (A, B) or C3HeB/FeJ (C, D) mice. Eight weeks following LDA infection, mice were treated for 3, 5, or 8 weeks with 150 (A, C) or 300 mg/kg (B, D) PZA via oral gavage. Pre-Rx represents untreated mice aerosol infected for 8 weeks. Ovals with solid lines represent the PZA less-responsive subpopulation, and dashed lines represent the PZA responsive subpopulation of C3HeB/FeJ mice. Pre-Rx and defined time points were determined to be statistically different (*; P < 0.01) using a one-way ANOVA. Responsive and less-responsive populations were determined to be statistically different (P < 0.001) using a two-way ANOVA.

Figure 2

BDQ exhibited dose-dependent activity between 10 and 25 mg/kg. Plots represent log10 CFU determinations from the lungs of individual BALB/c (A, B) or C3HeB/FeJ (C, D) mice. Eight weeks following LDA infection, mice were treated with BDQ 10 mg/kg (A, C) or 25 mg/kg (B, D) via oral gavage. BALB/c mice were treated for either 2 or 4 weeks. C3HeB/FeJ mice were treated for either 2, 4, or 8 weeks. Pre-Rx represents untreated mice aerosol infected for 8 weeks. Ovals with solid lines represent the BDQ less-responsive subpopulation, and dashed lines represent the BDQ responsive subpopulation of C3HeB/FeJ mice. Pre-Rx and defined time points were determined to be statistically different (*; P < 0.01) using a one-way ANOVA. Responsive and less-responsive populations were determined to be statistically different (P < 0.001) using a two-way ANOVA.

Figure 3

BDQ/PZA combination therapy had activity against both subpopulations in C3HeB/FeJ mice. Plots represent log10 CFU determinations from the lungs of individual BALB/c (A) or C3HeB/FeJ (B) mice. Pre-Rx represents untreated mice 8 weeks following aerosol infection. Mice were treated with BDQ 10 mg/kg and PZA 150 mg/kg in combination via oral gavage. BALB/c mice were treated for either 2 or 4 weeks. C3HeB/FeJ mice were treated for either 2, 4, or 8 weeks. Ovals with solid lines represent the less-responsive subpopulation, and dashed lines represent the responsive subpopulation of C3HeB/FeJ mice. Pre-Rx and defined time points were determined to be statistically different (*; P < 0.01) using a one-way ANOVA. Responsive and less-responsive populations were determined to be statistically different (P < 0.001) using a two-way ANOVA.

Figure 4

Plasma and lung tissue concentration–time profiles of PZA and POA in BALB/c and C3HeB/FeJ mice following a single 150 mg/kg PZA oral dose. Data points indicate mean values (n = 5 mice per time point) with SD error bars.

Figure 5

Plasma and lung tissue concentration–time profiles of BDQ and M2 in BALB/c and C3HeB/FeJ mice following a single 25 mg/kg BDQ oral dose. Data points indicate mean values (n = 5 mice per time point) with SD error bars.

Figure 6

Arithmetic mean and SD for BALB/c and C3HeB/FeJ plasma and lung tissue population PK parameters for PZA and POA. Panel A shows a comparison of absorption (ka), clearance (CL), and volume of distribution (V) parameters for PZA and POA in both mouse strains. Panel B shows a comparison of half-life (t1/2) and penetration (PC) coefficient for PZA and POA in both mouse strains. Histogram height represents the arithmetic mean, with error bars representing mean ± SD. The tissue equilibrium half-life was calculated as t1/2 = ln(2)/k, where k is the corresponding tissue penetration rate. All values were calculated from the corresponding GM and GSD listed in Supplemental Table 1.

Figure 7

Arithmetic mean and SD for BALB/c and C3HeB/FeJ plasma and lung tissue population PK parameters for BDQ and M2. Panel A shows a comparison of absorption (ka), clearance (CL), and volume of distribution (V) parameters for BDQ and M2 in both mouse strains. Panel B shows a comparison of half-life (t1/2) and penetration (PC) coefficient for BDQ and M2 in both mouse strains. Histogram height represents the arithmetic mean, with error bars representing mean ± SD. The tissue equilibrium half-life was calculated as t1/2 = ln(2)/k where k is the corresponding tissue penetration rate. All values were calculated from the corresponding GM and GSD listed in Supplemental Table 2.

Figure 8

MALDI-MS time course images of PZA and POA in BALB/c mice acquired 0.08–8 h following a single oral 150 mg/kg dose of PZA. Cellular, non-necrotizing lesions (type III) are outlined in white. Images depict a single lung lobe obtained from a representative mouse (n = 4 mice per time point). A serial section was stained with hematoxylin and eosin (H&E) for comparison.

Figure 9

MALDI-MS time course images of PZA and POA in C3HeB/FeJ mice acquired 0.08–8 h following a single oral 150 mg/kg dose of PZA. Caseous necrotic granulomas (type I) are outlined in white. Images depict a single lung lobe obtained from a representative mouse (n = 4 mice per time point). A serial section was stained with hematoxylin and eosin (H&E) for comparison.

Figure 10

MALDI-MS time course images of BDQ and M2 in BALB/c mice acquired 8–168 h following a single oral 25 mg/kg dose of BDQ. Cellular, non-necrotizing lesions (type III) are outlined in white. Images depict a single lung lobe obtained from a representative mouse (n = 4 mice per time point). A serial section was stained with hematoxylin and eosin (H&E) for comparison.

Figure 11

MALDI-MS time course images of BDQ and M2 in C3HeB/FeJ mice acquired 8–168 h following a single oral 25 mg/kg dose of BDQ. Caseous necrotic granulomas (type I) are outlined in white. Images depict a single lung lobe obtained from a representative mouse (n = 4 mice per time point). A serial section was stained with hematoxylin and eosin (H&E) for comparison.

Figure 12

Reduced penetration of BDQ into caseous necrotic granulomas. Light micrograph of a caseous necrotic granuloma (A; arrow) and a corresponding MALDI-MS image (B) acquired with a saturated intensity scale showing no detectable BDQ within the central caseum of the granuloma. Representative MALDI-MS image of a single lung lobe obtained from a C3HeB/FeJ mouse (n = 5). Mice were given a single 25 mg/kg oral dose of BDQ 1 h prior to necropsy.

Figure 13

Differential penetration of BDQ and M2 (N-desmethyl-BDQ) into caseous necrotic granulomas. MALDI-MS images of BDQ (red) and M2 (green) acquired 24 h (A) and 72 h (B) following a single oral 25 mg/kg dose of BDQ. Images represent a single lung lobe obtained from C3HeB/FeJ mice.

Figure 14

BDQ accumulated to a greater degree within THP-1 cells. THP-1 cells were exposed to 1 mM PZA, 0.4 mM POA, or 0.5 mM BDQ. Intracellular-to-extracellular ratios were calculated based upon LC/MS/MS quantification of drug levels following washing and cell lysis.