1 Optimization of the use of anti-TB drugs based on in situ pharmacokinetics Landry BLANC CNRS – CBMN, UMR R 52 5248 48, Uni niversi sité de de Bor ordeaux, CGFB RIC RICAI 2019 - 17 17 Déc écembre 2019 2019
Lung lesions due to tuberculosis Dartois V, Nat Rev Microbiol, 2014 2
Lung lesions due to tuberculosis Dartois V, Nat Rev Microbiol, 2014 3
Lung lesions due to tuberculosis Dartois V, Nat Rev Microbiol, 2014 4
Attenuation of antibiotic bactericidal activity on non-replicating bacteria Sarathy et al , Extreme Drug Tolerance of Mycobacterium tuberculosis in Caseum, AAC, 2018 90 in Anti tibiotic ic *M *MBC 90 Wayne Lob Lobel l Model Case Caseum 90 ( μ M) 90 ( μ M) rep eplicating mod odel MBC BC 90 M) MBC BC 90 M) 90 ( μ M) cult cu lture ( μ M) M) MBC BC 90 M) Rifampin 0.078 2 2 8 Isoniazid 0.31 – 0.63 >128 >128 >128 Pyrazinamide >80 >8,192 >8,192 512 Moxifloxacin 0.31 – 0.63 10 >128 2 Linezolid 10 >128 >128 128 Kanamycin 5.0 >128 80 >128 Clofazimine 40 >128 >128 >128 Bedaquiline 10 >20 >20 32 Rifapentine 0.078 0.5 10 2 Rifabutin 0.039 0.5 10 2 5 * Minimum Bactericidal Concentration
How to study and quantify drugs penetration in tuberculosis lesions ? • Lesions dissection coupled with LC-MS/MS • Laser Capture Microdissection (LCM) combined with LC-MS/MS • MALDI* Mass Spectrometry Imaging (MSI) 6 * * Matr trix ix-As Assis isted Laser Des esorptio tion/Ioniz izatio tion
Lesions dissection coupled with LC-MS/MS Chemical Homogenization Lesions dissection extraction and inactivation LC/MS/MS Quantification Fully quantitative Uninvolved lung tissues (UI) Tissue surrounding the lesions (SL), Cellular Lesions (LE), Highly sensitive and selective for the Wall of Cavitary lesions (CAW) drugs of interest Cavitary Caseum and Necrotic Center (CAC/NC). Dalin Rifat et al , STM, 2018 Poor spatial information Limited to the size of the original homogenized tissue 7
Laser Capture Microdissection (LCM) combined with LC-MS/MS Zimmerman & al, JOVE, 2018 Sn Snap frozen Sectionning Se sample Gamma irradiation Extrac acti tion Diss ssection LC LC/M /MS/MS Quan uanti tification 8
Laser Capture Microdissection (LCM) combined with LC-MS/MS Zimmerman & al, JOVE, 2018 Sn Snap frozen Se Sectionning sample Gamma irradiation Extrac acti tion Diss ssection LC LC/M /MS/MS Quan uanti tification • Fully quantitative capabilities • Spatially-resolved quantification • Blind dissection • Sensitivity of the LC/MS analysis 9
Ethambutol penetration in infected rabbit studied by LCM-LC/MS Zimmerman & al, AAC, 2017 * *Intramacrophagic Minimal Bacteriocidal Concentration • Steady state dosing : daily shot (100 mg/kg) during 1 week • Ethambutol reaches the required dose to elimate bacteria in each granuloma compartment 10
MALDI Mass Spectrometry Imaging (MSI): Principle Römpp et al ., Histochem. Cell Biol., 2013, 139, 759-789 • Regular MALDI mass spectrometry on each individual pixel (1 spectrum = 1pixel) • Multiple pixels acquisition allows to build a map for each detected peak 11 Mat atrix ix-Assis isted La Laser Des esorptio ion/Ioniz izatio ion
MALDI Mass Spectrometry Imaging: Workflow 3) Sample preparation 3a) Lipids, drugs metabolites, small molecules… 3b) Peptides/Proteins 3c) Glycans/Polysaccharides 2) Cryo-sectioning and thaw- mounting on slide 1) Snap frozen Gamma irradiated sample 4) Matrix application (sprayer / sublimation chamber) 8) Alignement with histological image 7) Data processing 5) AP-MALDI5 AF – Orbitrap 6) AP-MALDI5 AF – Orbitrap spectrum 12 High mas Hi ass re resolu lutio ion
High-resolution mapping of fluoroquinolones in TB rabbit lesions reveals specific distribution in immune cell types. L.Blanc et al , eLife, 2018 Uninvolved c lung Mac acrophages 2 mm N c Lymphocytes 2 mm MXF Foa oamy y Mac acrophages Caseum E Rabbit 1063 – 2h 2 mm LVX Penetration of fluoroquinolones in granuloma N seems following a pattern This pattern could correspond to layer of N different cell type Rabbit 2069 – 6h 2 mm GTX 13
Regions of Interest highlighting moxyfloxacin distribution defined by MALDI MSI and transposed on histological image L.Blanc et al , eLife, , 2018 14
Mathematical model of moxyfloxacin distribution to decipher key parameters driving it L.Blanc et al , eLife, , 2018 Computer 3 Key parameters : modeling MXF signal normalized to internal 0 .0 6 Distance from granuloma edge ↘ Di standard (pixel intensity) Percentage of macrophage ↗ 0 .0 4 Percentrage of necr necros osis is ↘ 0 .0 2 In vitro validation : drug uptake in different cell lines 0 .0 0 2 1 1 7 1 2 7 1 8 1 5 1 4 2 0 3 1 2 5 2 7 2 8 3 3 4 8 3 2 2 4 2 9 9 1 6 1 3 1 9 3 3 3 0 3 5 2 2 6 4 2 6 2 3 5 1 0 2 1 1 1 **** 30 * intracellular/extracellular * *** concentration ratio 25 * * 20 • Histopatholigical evaluation of cell *** * 15 populations in the different areas * 10 • % macrophage 5 • % lymphocyte 0 • % necrotique MXF LVX GTX • % neutrophile Macrophages Lymphocytes 15 Neutrophils Epithelial cells
Distribution study of clofazamine with high resolution MSI High spatial resolution with AP- SMALDI5-Orbitrap (TransMIT) Spatial resolution down to 5µm coupled with high resolution mass spectrometer 500 µm Pixel size down to cellular level Granuloma in infected mice Pixel size: 50 µm Treated daily with CFZ during 1 month Pixel size: 15 µm Very precise scan of clofazamine distribution Alignment of HE and MSI image to reveal clofazamine accumulation in macrophage Reaching foamy macrophage But limited penetration in necrotic core Pixel size: 5 µm 16
Take home message … TB Granuloma is a poorly vascularized region where drug penetration is complex Moreover, an extreme drug tolerance of Mycobacterium tuberculosis could be observed in caseum Cidal activity of most drugs is less efficient on non-replicating bacterias Drug exposure and MBC in situ are key factors Different mass spectrometry techniques are suitable for PK studies: LC/MS quantification on regular dissected tissue Sensitive and fully quantitative But limited in term of spatial information Laser Capture Microdissection coupled with LC/MS Sensitive and fully quantitative Investigation at granuloma structure level, but blind dissection Mass Spectrometry Imaging Less sensitive and semi quantitative Very valuable spatial information (cellular level) Combination of both techniques for LCM guided by Mass Spectrometry Imaging 17
Acknowledgment Nicolas Desbenoit Véronique Dartois Caroline Tokarski Brendan Prideaux 18
THANK YOU FOR YOUR ATTENTION 19
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