The clinical-stage drug BTZ-043 accumulates in murine tuberculosis lesions and efficiently acts against Mycobacterium tuberculosis

Abstract

The development of granulomas with central necrosis harboring Mycobacterium tuberculosis (Mtb) is the hallmark of human tuberculosis (TB). New anti-TB therapies need to effectively penetrate the cellular and necrotic compartments of these lesions and reach sufficient concentrations to eliminate Mtb. BTZ-043 is a novel antibiotic showing good bactericidal activity in humans in a phase IIa trial. Here, we report on lesional BTZ-043 concentrations severalfold above the minimal-inhibitory-concentration and the substantial local efficacy of BTZ-043 in interleukin-13-overexpressing mice, which mimic human TB pathology of granuloma necrosis. High-resolution MALDI imaging further reveals that BTZ-043 diffuses and accumulates in the cellular compartment, and fully penetrates the necrotic center. This is the first study that visualizes an efficient penetration and accumulation of a clinical-stage TB drug in human-like centrally necrotizing granulomas and that also determines its lesional activity. Our results most likely predict a substantial bactericidal effect of BTZ-043 at these hard-to-reach sites in TB patients.

Fig. 1. Efficacy of BTZ-043 in Mtb-infected BALB/c mice.

a and b BTZ-043 dose escalation study in the chronic model of TB. Female BALB/c mice were infected by aerosol with an average of 171 CFU Mtb H37Rv 3 weeks before treatment (−3). The pulmonary bacterial burden was assessed at the start of therapy (0) and after 4, 6 and 8 weeks of treatment with the indicated doses [mg/kg/day] of microcrystalline BTZ-043 (blue colors), while isoniazid (INH, 25 mg/kg/day, orange color) and vehicle (NT, white color) served as controls. Results are expressed as mean and SD (n = 5 mice per group for time points (-3), (0) and 8 weeks of treatment with 50 mg/kg BTZ-043, n = 6 mice for all other drug treatment groups; two-way ANOVA with Bonferroni’s posttest). c BTZ-043 dose-fractionation study. Female BALB/c mice were infected with an average of 20 CFU Mtb H37Rv 3 weeks before treatment started with different doses [mg/kg] of amorphous BTZ-043 (purple bars) administered either once (QD, filled bars) or twice (BID, open bars) daily or with microcrystalline BTZ-043 (QD, blue bar). INH therapy (10 mg/kg/day, light orange bar) or untreated mice (NT, white bar) served as controls. Treatment efficacy was determined after 6 weeks of therapy. Data represent mean and SD (n = 3 mice per group for the time point (0), the NT group and the 10 mg/kg BTZ-043 QD treatment group, n = 4 mice for all other treatment groups; one-way ANOVA with Bonferroni’s posttest for comparison of same total doses administered either BID or QD). Source data are provided as Source Data file.

Fig. 2. Pharmacokinetic profile of BTZ-043_total in mice.

Six naïve BALB/c mice per group were treated with amorphous formulation (*) or microcrystalline formulation (**) of BTZ-043 at indicated doses [mg/kg] for 5 days by oral gavage. a Plasma concentration-time curves (mean and SD, n = 3 mice per timepoint). b PK parameters (mean, n = 3 mice per timepoint). Source data are provided as Source Data file and as Supplementary Data 1.

Fig. 3. Early antimycobacterial activity of BTZ-043 under conditions of centrally necrotizing granulomas.

a Bacterial burden in lungs of IL-13^tg mice. Animals were infected with an average of 263 CFU Mtb H37Rv via the aerosol route. Nine weeks after infection, mice were treated with a daily dose of 250 mg/kg of BTZ-043 for 10 days. The pulmonary bacterial burden was determined before and after therapy (8.55 log₁₀ CFU and 7.69 log₁₀ CFU, respectively). Data represent mean and SD (n = 4 mice per group before therapy, n = 7 mice per group for BTZ-043 treatment) and statistical analysis was performed by an unpaired student’s t test (two-tailed) on log₁₀-transformed CFU data. One experiment representative of 2 is shown. Source data are provided as Source Data file. b Detection of acid fast bacilli in centrally necrotizing granulomas of untreated or BTZ-043 treated mice by Ziehl-Neelsen (ZN) staining. Animals were infected by aerosol with an average of 106 CFU Mtb H37Rv and treatment with 250 mg/kg/day of BTZ-043 for 10 days started 9 weeks after infection. Upper panels: higher magnifications of ZN-stained specimens with rimmed zones of foamy macrophages. Total pulmonary CFU counts determined for the respective mouse are shown in the lower left corner. Middle panels: lower magnifications of the same ZN-stained specimens depicting the entire granulomas and areas of higher magnification displayed in the upper panels are outlined. Lower panels: subsequent CD68/ZN staining of the same sections for the detection of foamy macrophages. Images are representative of 2 independent experiments. Scale bars: upper panel 50 µm, middle and lower panel 200 µm.

Fig. 4. Pulmonary and lesional efficacy of BTZ-043 over the course of treatment.

Animals were infected with an average of 341 CFU Mtb H37Rv via the aerosol route. Ten weeks after infection, mice were treated 5 times a week with 250 mg/kg of BTZ-043 or the vehicle control. After 2 weeks and between 4 and 6 weeks, lungs were removed to determine the bacterial load in the lung and in the necrotic center of granulomas. Since some mice receiving the vehicle suspension became moribund, the second analysis time point comprises a treatment period of 4 to 6 weeks. a Pulmonary bacterial burden determined by CFU counts on solid agar medium. Data represent mean and SD (n = 5 mice per group for BTZ-043 treatment groups and the group receiving vehicle for 2 weeks, n = 6 mice for the group receiving vehicle for 4 to 6 weeks), and statistical analysis was performed by an unpaired student’s t test (two tailed) on log₁₀-transformed CFU data. b Lesional efficacy of BTZ-043. Left panel: Visual representation of necrotic area collected by LCM. Scale bar: 200 µm. Right panel: The local bacterial load was quantified by 16S rRNA gene expression of Mtb using a molecular bacterial load assay (MBLA) after LCM of the necrotic core of granulomas on serial lung sections normalized to the volume of the isolated area. Data represent mean and SD (n = 2 mice per group for 2 weeks of therapy, n = 3 mice per group for 4 to 6 weeks of therapy; two experiments were conducted: 1 experiment with an analysis time point after 2 weeks of treatment and 1 repeat experiment in which samples were obtained 2 weeks and between 4 and 6 weeks of treatment; the results of the local bacterial loads in the two experiments were similar). Source data are provided as Source Data file.

Fig. 5. Correlation of lesion pathology and BTZ-043 penetration.

Treatment with 250 mg/kg/day of BTZ-043 for 10 days started 9 weeks after infection with 263 CFU Mtb H37Rv. Lung tissue of an IL-13^tg mouse was collected 2 h after the last administration and serial cryosections were prepared for histological characterization of lesions and MALDI imaging. a HE-stained specimen. b Trichrome-stained specimen. c CD68/ZN-stained specimen. d Oil Red O-stained specimen. e Ion image of BTZ-043 [M + H]⁺ C₁₇H₁₇F₃N₃O₅S⁺, m/z 432.08355, 10 µm pixel size. The blue and purple lines indicate the determined granuloma edges. The stainings given in a, b and c were performed on sections directly neighboring to the one used for MALDI imaging, while the Oil Red O-stained specimen is 16 tissue sections (corresponding to 192 µm) away. Histological images are representative of 2 biological replicates. Scale bars: a-e: 1 mm. f Penetration plots of BTZ-043 into both granulomas (Fig. 5e) present in the section. Black vertical lines indicate the edges of the granulomas. Gray areas inside the granulomas indicate the cellular regions, which mainly consist of foamy macrophages, and necrotic areas. Source data are provided as Source Data file and as Supplementary Data 2 and 8.

Fig. 6. Pulmonary BTZ-043 concentrations.

IL-13^tg mice were infected with 263 CFU Mtb H37Rv and after 9 weeks, animals were treated with 250 mg/kg/day of BTZ-043 for 10 days prior to lung tissue collection at indicated time points after the last administration. a Change of pulmonary BTZ-043 concentration over time. Lung cryosections (n = 5) from 1 mouse per time point were prepared for BTZ-043 quantification by LC-MS/MS which is depicted as [ng/mg] on the left y axis. The right y axis shows the corresponding BTZ-043 concentrations [ng/mL] calculated based on an assumed tissue density of 1 g/mL. Data are shown as mean and SD. b BTZ-043 concentrations in necrotic granulomas and non-necrotic lung tissue 4 h after administration. Left panel: HE-stained lung section to visualize necrotic granulomas and non-necrotic regions after sampling by LCM. Scale bar: 1 mm. Right panel: BTZ-043 concentrations determined in necrotic granulomas and non-necrotic regions by coupling LCM and LC-MS/MS which is depicted as [ng/mg] on the left y axis. The right y axis shows the corresponding BTZ-043 concentrations [ng/mL] calculated based on an assumed tissue density of 1 g/mL. Lung cryosections obtained from 1 mouse were used to collect 3 samples of each tissue type for subsequent LC-MS/MS measurements. Data represent mean and SD (n = 3; color code: data points with same color indicate LCM sampling from same cryosections). Source data are provided as Source Data file and as Supplementary Data 3 and 4.

Fig. 7. Spatial and temporal distribution of BTZ-043 in centrally necrotizing granulomas of IL-13^tg mice by MALDI imaging.

Mice were infected with 263 CFU Mtb H37Rv and treatment with 250 mg/kg/day of BTZ-043 for 10 days started 9 weeks after infection. Lung tissue was collected 0.5 h (a, e), 2 h (b, f), 4 h (c, g) and 8 h (d, h) after the last administration. a–d Post-measurement HE staining. e–h Distribution of BTZ-043 [M + H]⁺ in these sections by MALDI imaging, pixel size 30 µm. Maximum intensities are given at the bottom of each image. Histological images are representative of 2 biological replicates. Scale bars: 1 mm. Source data are provided as Supplementary Data 2 and 8.