Mice. Female BALB/c mice were purchased from JANVIER LABS (Le Genest Saint-Isle, France) or Charles River Laboratories (Sulzfeld Germany, or Wilmington, MA, USA). IL‑13tg mice expressing murine il13 under the control of the human CD2 locus control region45 [Tg(CD4-Il13)431Anjm] backcrossed to the BALB/c genetic background were bred under specific-pathogen-free conditions at the Christian-Albrechts-University of Kiel (Kiel, Germany) or the Max-Planck-Institute for Evolutionary Anthropology (Leipzig, Germany). Mtb-infected mice were maintained in individually ventilated cages (IVC, Ebeco, Castrop-Rauxel, Germany) under biosafety level 3 (BSL-3) conditions at the Research Center Borstel (RCB). Female BALB/c mice receiving drug treatment without prior infection were housed under standard conditions at the RCB. Animal experimentation conducted at the RCB was in accordance with the German regulations of the Society for Laboratory Animal Science (GV-SOLAS) and the European Health Law of the Federation of Laboratory Animal Science Associations (FELASA). All animal experiments were approved by the animal research ethics committee of the federal state of Schleswig-Holstein prior to permission by the Ministry of Energy, Agriculture, the Environment, Nature, and Digitalization (Kiel, Germany; permits 3-1/15, 69-6/16, and 84-9/20). For experiments performed at Johns Hopkins University all procedures involving the care and use of animals in the study were reviewed and approved by the Johns Hopkins University Animal Care and Use Committee (protocol #MO15M479). The care and use of animals were conducted in accordance with the principles outlined in the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), the Animal Welfare Act, the American Veterinary Medical Association (AVMA) Euthanasia Panel on Euthanasia, and the Institute for Laboratory Animal Research (ILAR) Guide to the Care and Use of Laboratory Animals.
BTZ‑043 dose escalation in Mtb-infected BALB/c mice. Aerogenic infection of female BALB/c mice with an intermediate dose of Mtb H37Rv was performed in a Glas-Col inhalation exposure system as previously described46. Five mice were sacrificed the following day to determine the number of bacteria implanted in the lung and 3 weeks later to determine the pulmonary bacterial burden at the start of treatment (n=5). BTZ‑043 was formulated every 3 weeks by suspending BTZ‑043 (microcrystalline, containing 1.8% Tween 80) in 1% CMC sodium salt and Tween 80 added to a final concentration of 0.5%. BTZ‑043 suspensions were stored at 4 °C and, prior to use, acclimated to room temperature and mixed to achieve homogenous suspensions before administration. INH was dissolved in distilled water, stored at 4 °C and prepared weekly. Treatment started 3 weeks after infection and drugs were administered by oral gavage 5 days per week with daily doses of 50, 100, 250, 500 or 1,000 mg/kg body weight for BTZ‑043, 25 mg/kg body weight for INH or CMC/Tween 80 as vehicle control. After 4, 6 and 8 weeks of treatment 5 to 6 mice per therapy group were sacrificed, lungs were aseptically removed, weighed, and homogenized. Tenfold serial dilutions of lung homogenates were plated on Middlebrook 7H10 agar supplemented with 10% bovine serum and incubated at 37 °C for 3 weeks. Colonies on plates were enumerated and results expressed as log10 CFU per lung for the comparison of treatment efficacy.
BTZ‑043 dose-fractionation in Mtb-infected BALB/c mice. Female BALB/c mice, 8-10 weeks old, were aerosol-infected using a Glas-Col inhalation exposure system and a thawed aliquot of broth culture having a known bacterial titer 3 weeks prior to treatment. Mice were sacrificed for lung CFU counts the following day (n=3) and 3 weeks later at the initiation of treatment (n=3) to determine the number of CFU implanted and the number present at the start of treatment, respectively. Both, BTZ‑043 microcystalline (containing 1.8% Tween 80) and BTZ‑043 amorphous (hot melt extrudate BTZ‑043 / Soluplus 1:5) particles were suspended in 1% CMC sodium salt and Tween 80 added to a final concentration of 0.5%. On weekly basis, INH was dissolved in distilled water and kept refrigerated at 4°C. The untreated control group received no treatment. The BTZ‑043 formulations were mixed by shaking before administration. Treatment started 3 weeks after infection. Drugs were administered 5 days per week, by gavage, either once or twice daily. For all BID dosing, the time between administrations was 7 - 8 h. The treatment scheme is presented in the Supplementary Table 4. After 6 weeks of treatment, 4 mice per treatment group were sacrificed, lungs were aseptically removed and homogenized in glass grinders. Serial 10-fold dilutions of the homogenates were prepared, and 0.5 ml aliquots were plated on 7H11 agar plates47. Baseline lung log10 CFU counts were assessed the day after aerosol infection (day -20) and at treatment initiation (day 0). Treatment efficacy was assessed by comparing lung log10 CFU counts after 6 weeks of treatment.
PK investigation. Naïve BALB/c mice were dosed orally for 5 days with BTZ‑043 at doses used in the BTZ‑043 dose-fractionation study in Mtb-infected BALB/c mice (2.5, 5, 50, 250 mg/kg/day) to reach steady state (Study no. 832.220.5501). On day 5, plasma samples were collected for bioanalytical analysis pre-dose and at 0.5 h, 1 h, 2 h, 4 h and 8 h post-dosing (see Supplementary Table 1 for more details).
BTZ‑043 partially forms the unstable metabolite M2 in vivo, a hydride Meisenheimer complex48. This metabolite can only be quantified indirectly by reverse conversion to the parent compound. The amount of BTZ‑043 measured after reverse conversion of M2 is called BTZ‑043total. In an amber 0.5 ml Eppendorf vial, 20 µL of plasma were mixed with 2 µL of methanol (MeOH). 10 µL of formic acid (FA; Suprapur, Merck, Darmstadt, Germany) in MilliQ Water (50% (v/v) were added and the samples mixed by vortexing. Afterwards, the samples were mixed for additional 60 min at 40 °C in a thermoshaker. Protein precipitation at room temperature followed with 60 µL acetonitrile (ACN)/internal standard. Samples were mixed by vortexing and after centrifugation (9,727 x g / 10 min / room temperature) supernatants were transferred into an amber autoinjector vial (0.4 mL). LC was performed on an Agilent 1100 Series HPLC (Agilent Technologies, Santa Clara, CA, USA) using a Inertsil ODS-4 C18 column (GL Sciences Inc. 2.1 x 75 mm, 3 µm particle size) with an Inertsil ODS-4 guard column (1.5 x 10 mm, 3 µm particle size) at a column temperature of 25 °C. Mobile phase solvent A: 0.1% FA. Solvent B: MeOH with 0.1% FA. The gradient started at 50% B and was increased to 75% until minute 1 which was held until minute 3. B was raised to 100% at 3.1 minutes and held until minute 6. At 6.1 min B was dropped to 50% and held until minute 10 resulting in a total runtime of 10 minutes. Injection volume was 1 µL at high concentration (20 – 7,000 ng/mL) and 2 µL at low concentration levels (5 – 2,000 ng/mL). Autosampler temperature was set at 5 °C. Electrospray ionization (ESI) multiple reaction monitoring (MRM) MS/MS on an API 2000 (Applied Biosystems, Waltham, MA, USA) triple quadrupole mass spectrometer was used for signal detection. The transition 432.1 à 82.9 in positive ion mode was used for quantification. Spray voltage was 4.5 kV and source temperature 400°C. Data analysis was performed in Analyst 1.4.2.
Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. An in vitro permeability assay with Caco-2 cells was performed according to a standard protocol28. Briefly, Caco-2 cells (DSMZ no.: ACC 169) were grown on permeable inserts (Corning® 12-well Transwell with 0.4 μm pore polyester membrane insert (diameter of 12 mm) from Merck, Darmstadt, Germany) for 3 weeks to obtain a monolayer of polarized Caco-2 cells. The apical and basolateral compartment contained a volume of 0.4 mL and 1.2 mL, respectively. The permeation of BTZ‑043 and PZA was monitored from apical to the basolateral compartment and vice versa by taking samples from the receiver compartment after different time points (0, 10, 30, 60, 90 and 120 min). Atenolol (reference compound for low permeability) and propranolol (reference compound for high permeability) were used as controls. All experiments were performed in triplicates for each transport condition. Prior and subsequent to the incubation period, each insert was checked for integrity and tightness of the Caco-2 monolayer by measuring the transepithelial electrical resistance (TEER) with Epithelial Volt/Ohm Meter 3 (EVOM3, Word Precision Instrument, Sarasota, FL, USA). Furthermore, the integrity of the Caco-2 monolayer after to the permeation experiment was checked through addition of Lucifer Yellow to the apical compartment and incubation for 1 h. The extent of permeation (%) was determined by fluorescence measurement using a SPARK platereader (Tecan, Grödig, Austria). The subsequent quantification of drugs is described in the supplementary information section.
BTZ‑043 treatment of Mtb-infected IL‑13tg mice. Aerogenic infection of IL‑13tg mice with an intermediate dose of Mtb H37Rv using a Glas-Col inhalation exposure system was performed as previously described46. Five mice were sacrificed the following day to determine the number of bacteria implanted in the lung. After 9 weeks of infection the development of centrally necrotizing granulomas was histologically confirmed, and the pulmonary bacterial burden was determined (n=4). Mice received 10 daily doses of 250 mg/kg BTZ‑043 by oral gavage to reach steady state conditions and were euthanized at defined time points (0.5 h, 2 h, 4 h, 8 h) after the last administration. Lungs were aseptically removed and tissue containing macroscopically visible lesions was snap frozen in liquid nitrogen for subsequent cryosectioning. To assess the bacterial burden, the remaining lung tissue was homogenized, tenfold serial dilutions of organ homogenates were plated on Middlebrook 7H10 agar supplemented with 10% bovine serum and incubated at 37 °C for 3 weeks. Colonies on plates were enumerated and results expressed as log10 CFU per lung for the comparison with baseline lung log10 CFU counts determined before the start of treatment.
Histology and immunohistochemistry. Histopathological evaluation, ZN staining, immunohistochemical analysis of CD68 in combination with ZN staining, detection of collagen deposition, and lipid droplets were performed as previously described10,13. Images were acquired on a BX41 light microscope (Olympus, Hamburg, Germany) using cellSens imaging software (Olympus, Hamburg, Germany) or NIS-Elements software (Nikon, Badhoevedorp, The Netherlands). Post MALDI imaging haematoxylin and eosin staining was performed as described previously49.
Tissue cryosectioning and irradiation. Serial cryosections (12 µm) were cut from unembedded lung tissue at -25 °C using a Leica CM1850 or CM3050s cryostat (Leica Microsystems, Wetzlar, Germany) and thaw mounted onto adhesive glass slides (SuperFrost™) as described by Treu et al. 49. Sections were stored at -80 °C until further processing. Cryosections were g-irradiated (5.85 kGy, BIOBEAM 8000, Gamma-Service Medical GmbH, Leipzig, Germany) on dry ice as described previously13.
LC-MS/MS of tissue sections. Extraction procedure for lung cryosections: Prior to sample preparation for LC-MS/MS measurements, the weight of cryosections was determined as described previously13. After drying of lung cryosections in a SpeedVac, samples were reconstituted with 25 µL LC-MS-grade water, 200 µL ACN (reserpine concentration: 12.5 ng/mL) and 25 µL 1% FA. Samples were vortexed and afterwards centrifuged for 10 min at 15,000 × g, room temperature. Approximately 200 µL of the resulting supernatant were transferred in a 1.5 mL Eppendorf tube and re-centrifuged under the same conditions. Quantification by LC-MS/MS: Liquid chromatography was performed on an Agilent 1100 Series HPLC (Agilent Technologies, Santa Clara, CA, USA) using a SeQuant® ZIC®-HILIC column (Merck Millipore SeQuant, 2.1 inner diameter x 150 mm length with 5 µm particle size, pore-size 200 Å) at a column temperature of 30 °C. The mobile phase consisted of 1% FA (solvent A) and ACN (solvent B). The gradient started at 90% B at a flowrate of 0.5 mL/min. After 1 minute of isocratic conditions, the percentage of ACN was decreased to 2% B until minute 4. At minute 4, the flowrate was increased to 0.8 mL/min. The gradient was kept isocratic at 2% B with a flowrate of 0.8 mL/min for 6 minutes until minute 10. Afterwards, the percentage of ACN was re-increased to 90% B until minute 15 and the flowrate re-decreased to 0.5 mL/min after minute 19. These conditions were maintained for 1 minute, so that the total run time was 20 minutes. The autosampler temperature was set to 4 °C and sample injection volume was 5 µL. The Waters Micromass Quattro Premier XE triple quadrupole mass spectrometer (Waters Corporation, Milford, MA, USA) using ESI was operated in positive ion mode using MRM. The transition 432.1 à 292.3 of BTZ‑043 (cone voltage 30 EV, collision energy 30 EV, dwell time 0.1 s) was used as the quantifier. The reserpine transition 608.6 à 194.9 (cone voltage 30 EV, collision energy 35 EV, dwell time 0.1 s) was used as the internal standard. The cone gas- and desolvation gas flow were set to 100 L/h and 800 L/h, respectively. The extractor voltage was 3.0 V. We optimized the capillary voltage, the source-, and the desolvation gas temperature and chose 3.0 kV, 90 °C, and 450 °C, respectively. MassLynx 4.1 and TargetLynx (Waters Corporation, Milford, MA, USA) were used for operating the platform and quantifying the samples, respectively.
BTZ‑043 calibration curve for quantification. Lung homogenate prepared from naïve mice was dried in a SpeedVac and reconstituted in 100 µL LC-MS-grade water. Then 800 µL ACN, containing reserpine (12.5 ng/mL per sample) as reference for quantification and ionization, and 100 µL 1% FA were added. The solution was incubated for 10 min at room temperature with continuous shaking at 1300 rpm. To avoid floating particles, the solution was centrifuged for 10 min at 15,000 × g. The resulting supernatant was collected in a separate tube and was once more centrifuged under the same conditions (10 min, 15,000 × g). Calibration BTZ‑043 standard was prepared by diluting a stock solution in extracted lung tissue homogenate with a curve range as 0.001 – 0.25 µg/mL.
MALDI imaging and penetration analysis. Lung cryosections were shipped on dry ice from the RCB to the University of Bayreuth for MALDI imaging analysis and stored at -80°C. Sections were brought to room temperature inside a desiccator for 15 min. DCTB matrix (5 mg/mL, 1:1 chloroform/ethanol acidified with 0.1 Vol.% trifluoroacetic acid) was applied using a pneumatic sprayer system built in house. Deuterated standard BTZ‑043 D4 was applied (0.5 μg/mL in 1:1 acetone/water total applied volume 25 μL) using a pneumatic sprayer built in house. Imaging measurements were performed using an AP-SMALDI 10 (TransMIT GmbH, Gießen, Germany) atmospheric pressure MALDI imaging source equipped with a λ = 337 nm N2 laser operating at repetition rate of 60 Hz coupled to a Q Exactive HF (Thermo Fisher Scientific, Bremen, Germany) orbital trapping mass spectrometer. Measurements were conducted with 30 laser shots per pixel and a mass range of m/z 420-540 with a step size of 10 x 10 µm or 30 x 30 µm. Ion images and RGB overlays were generated in MSiReader Version 1.050 with a bin width of 2 ppm. In order to assess possible artifacts as a result of different tissues properties in granuloma and surrounding tissue, an isotopically labeled BTZ‑043 standard was applied in an additional application step. This experiment showed that the detected BTZ‑043 signal did not depend on the tissue properties and therefore ion suppression effects can be ruled out as a source of relevant artifacts. (see Supplementary Fig. 9 for more details). Penetration plots were generated using our previously reported penetration analysis tool12. The generation of the granuloma edge was based on the co-detected ion m/z 482.36050 highlighting the granuloma areas. To determine the on tissue region, the co-detected ion m/z 488.44620 was used. See the Supplementary Fig. 7 for single ion images. All measurements were conducted using the matrix ion m/z 539.25715 [2M+K]+ as the reference for internal mass calibration49. Mass accuracies across imaging datasets are given as the root mean square error (RMSE) of the δm/z values in ppm of all pixels containing the targeted ion within a ±3 ppm window of the theoretical m/z. The Supplementary Table 5 gives the RMSE values of BTZ‑043 in measurements included in this study. MS/MS experiments were conducted with a precursor isolation window of ± 0.2 m/z using higher energy collision induced dissociation (HCD 26).
Statistical analysis. If applicable, statistical analysis was performed using Prism 9 (Graphpad Software, San Diego, CA, USA). Quantifiable data are expressed as the means of individual determinations and standard deviations (SD). Log-transformed CFU data were evaluated by Student’s t-test, by a one-way or two-way ANOVA followed by Bonferroni`s post hoc test for multiple comparisons. BTZ‑043 concentrations were tested for normality and analyzed by one-way ANOVA followed by Tukey`s post hoc test for multiple comparisons.