Optimization of the use of anti-TB drugs based on in situ - - PowerPoint PPT Presentation
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 sit de de Bor ordeaux, CGFB RIC RICAI 2019 - 17 17 Dc cembre 2019 2019 Lung lesions due to
Landry BLANC
CNRS – CBMN, UMR R 52 5248 48, Uni niversi sité de de Bor
RIC RICAI 2019 - 17 17 Déc écembre 2019 2019
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Lung lesions due to tuberculosis
Dartois V, Nat Rev Microbiol, 2014
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Lung lesions due to tuberculosis
Dartois V, Nat Rev Microbiol, 2014
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Lung lesions due to tuberculosis
Dartois V, Nat Rev Microbiol, 2014
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Attenuation of antibiotic bactericidal activity on non-replicating bacteria
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Anti tibiotic ic *M *MBC90
90 in
rep eplicating cu cult lture (μM) M) Wayne mod
MBC BC90
90 (μM)
M) Lob Lobel l Model MBC BC90
90 (μM)
M) Case Caseum MBC BC90
90 (μM)
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
* Minimum Bactericidal Concentration
Sarathy et al, Extreme Drug Tolerance of Mycobacterium tuberculosis in Caseum, AAC, 2018
How to study and quantify drugs penetration in tuberculosis lesions ?
with LC-MS/MS
6 * * Matr trix ix-As Assis isted Laser Des esorptio tion/Ioniz izatio tion
Lesions dissection coupled with LC-MS/MS
Fully quantitative Highly sensitive and selective for the drugs of interest Poor spatial information
Limited to the size of the original homogenized tissue
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Lesions dissection Homogenization Chemical extraction and inactivation
Uninvolved lung tissues (UI) Tissue surrounding the lesions (SL), Cellular Lesions (LE), Wall of Cavitary lesions (CAW) Cavitary Caseum and Necrotic Center (CAC/NC).
Dalin Rifat et al, STM, 2018
LC/MS/MS Quantification
Laser Capture Microdissection (LCM) combined with LC-MS/MS
8 Zimmerman & al, JOVE, 2018
Sn Snap frozen sample Gamma irradiation Se Sectionning Extrac acti tion LC LC/M /MS/MS Quan uanti tification Diss ssection
Laser Capture Microdissection (LCM) combined with LC-MS/MS
9 Zimmerman & al, JOVE, 2018
Sn Snap frozen sample Gamma irradiation Se Sectionning Extrac acti tion LC LC/M /MS/MS Quan uanti tification Diss ssection
Ethambutol penetration in infected rabbit studied by LCM-LC/MS
granuloma compartment
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*
*Intramacrophagic Minimal Bacteriocidal Concentration
Zimmerman & al, AAC, 2017
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Römpp et al., Histochem. Cell Biol., 2013, 139, 759-789
MALDI Mass Spectrometry Imaging (MSI): Principle
Mat atrix ix-Assis isted La Laser Des esorptio ion/Ioniz izatio ion
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MALDI Mass Spectrometry Imaging: Workflow
1) Snap frozen Gamma irradiated sample 2) Cryo-sectioning and thaw- mounting on slide 4) Matrix application (sprayer / sublimation chamber) 5) AP-MALDI5 AF – Orbitrap Hi High mas ass re resolu lutio ion 8) Alignement with histological image 7) Data processing 6) AP-MALDI5 AF – Orbitrap spectrum 3) Sample preparation 3a) Lipids, drugs metabolites, small molecules… 3b) Peptides/Proteins 3c) Glycans/Polysaccharides
High-resolution mapping of fluoroquinolones in TB rabbit lesions reveals specific distribution in immune cell types.
Penetration of fluoroquinolones in granuloma seems following a pattern
This pattern could correspond to layer of different cell type
Rabbit 1063 – 2h Rabbit 2069 – 6h
MXF LVX GTX
2 mm 2 mm 2 mm
N N N
E
Uninvolved lung Mac acrophages Lymphocytes Foa
y Mac acrophages Caseum
c
2 mm
c
13 L.Blanc et al, eLife, 2018
Regions of Interest highlighting moxyfloxacin distribution defined by MALDI MSI and transposed on histological image
14 L.Blanc et al, eLife, , 2018
Mathematical model of moxyfloxacin distribution to decipher key parameters driving it
populations in the different areas
5 10 15 20 25 30 MXF LVX GTX
intracellular/extracellular concentration ratio
Macrophages Lymphocytes Neutrophils Epithelial cells **** * * *** * * *** * *
In vitro validation : drug uptake in different cell lines
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 0 .0 0 0 .0 2 0 .0 4 0 .0 6
MXF signal normalized to internal standard (pixel intensity)
Computer modeling
3 Key parameters : Di Distance from granuloma edge ↘ Percentage of macrophage ↗ Percentrage of necr necros
is ↘
15 L.Blanc et al, eLife, , 2018
Distribution study of clofazamine with high resolution MSI
500 µm
Pixel size: 50 µm Pixel size: 15 µm Pixel size: 5 µm
High spatial resolution with AP- SMALDI5-Orbitrap (TransMIT) Spatial resolution down to 5µm coupled with high resolution mass spectrometer
Pixel size down to cellular level
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
Granuloma in infected mice Treated daily with CFZ during 1 month
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TB Granuloma is a poorly vascularized region where drug penetration is complex Moreover, an extreme drug tolerance of Mycobacterium tuberculosis could be
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
Take home message…
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Acknowledgment
Véronique Dartois Nicolas Desbenoit Caroline Tokarski Brendan Prideaux
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