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Applications of Quantitative Mass

Spectrometry Imaging in drug development

David BONNEL, PhD ImaBiotech, France

EBF 7th Open Meeting, Barcelona, 2014

© ImaBiotech 2014

m/z Int.

X1, Y1

m/z Int.

X2, Y1

m/z Int.

X3, Y1

m/z Int.

X4, Y1

m/z Int.

X4, Y2

m/z Int.

X3, Y2

m/z Int.

X2, Y2

‘Nb of pixels’ = ‘nb of mass spectra’

Mass Spectrometry Imaging (MSI)?

© ImaBiotech 2014

m/z Int.

X1, Y1

m/z Int.

X2, Y1

m/z Int.

X3, Y1

m/z Int.

X4, Y1

m/z Int.

X4, Y2

m/z Int.

X3, Y2

m/z

Int.

Each m/z signal = One specific MS image

Mass Spectrometry Imaging (MSI)?

© ImaBiotech 2014

WB Clozapine and its related metabolites distributions using MSI

Distribution of endogenous metabolites

Mass Spectrometry Imaging (MSI)?

© ImaBiotech 2014

Distribution of endogenous metabolites

Mass Spectrometry Imaging (MSI)?

©2014 ImaBiotech

Fibrotic lung tissue

MSI and Histology

Glomeruli of kidney

Brain section

Amyloid plaques with Red Congo

Oil red O staining on skin tissue

MSI is a complementary technique to support histology

©2014 ImaBiotech

Bioanalytical Problematics

ANALYTICAL Variability

MALDI Matrix deposition

Mass Spectrometer deviation

BIOLOGICAL Variability

o Inter-/intra- tissue variation

o Ion suppression due to tissue/Ionization Yield

CALIBRATION CURVE

CC with criteria

LOD/LOQ Calculation

Absolute Quantification

Relative Quantification

©2014 ImaBiotech

QMSI workflow

Tissue

Section

Matrix

MSI

Process.

D Biological

D Analytical

D Analytical

D Analytical

CC Criteria

Dilution Range

« Pseudo » Internal Standard

Dosed Tissue

+

-

+

-

Quality Control

©2014 ImaBiotech

Applications ILC Small Molecule

Target molecule Diazepam

Model Dosed Rat (15 mg/kg, i.v.,

15 min)

Sample Brain

Therapeutic area Anxiolytic, anticonvulsant

Preparation Cryosection (10 µm)

Matrix DHB

ILC d5-Diazepam

(30 µM, m/z 290)

Ion images [M+H]+, m/z 285

Raster size 200 µm

Applications of QMSI

Instrumentation: MALDI-TOF Mass Spectrometer Autoflex Speed (Bruker Daltonics) Matrix Automated Deposition Device SunCollect (SunChrom)

©2014 ImaBiotech

Biological variability: Ion suppression on tissue

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Ventricle White Matter Hippocampus Thalamus Cortex

TEC

Val

ue

s

Distribution of d5-Diazepam deposited on the brain & on the slide (200 µm)

Evaluate the specific ion suppression of each kind of tissues Tissue type specific Molecule specific

TEC = Int (Tissue) Int (Slide)

< 1

m/z 290.15

Image from QuantinetixTM

In triplicate

Reference Signal Cortex

Hippocampus

Ventricle

White Matter

Thalamus

©2014 ImaBiotech

Histological tissue effect: Intra-Normalization & “Real” Image

Normalization of molecular image using ILC

See the “real” distribution of the analyte1

Normalized image/d5-Diazepam Unnormalized image

m/z 285.06

Image from QuantinetixTM

Distribution of Diazepam within dosed brain section (200 µm)

©2014 ImaBiotech

Histological tissue effect: Intra-Normalization & “Real” Image

Normalization of molecular image using ILC

See the “real” distribution of the analyte1

Normalized image/Diazepam-d5 Unnormalized image

m/z 285.06

Image from QuantinetixTM

Distribution of Diazepam within dosed brain section (200 µm)

0.0

0.5

1.0

1.5

4x10

Inte

ns. [a

.u.]

200 300 400 500 600 700 800

0

100

200

300

Inte

ns. [a

.u.]

283.5 284.0 284.5 285.0 285.5 286.0 286.5 287.0

Diazepam [M+H]+

m/z 285.06 S/N: 9

0.0

0.5

1.0

1.5

2.0

4x10

Inte

ns. [a

.u.]

200 300 400 500 600 700 800

0

100

200

300

Inte

ns. [a

.u.]

283.5 284.0 284.5 285.0 285.5 286.0 286.5 287.0

Corronal Section Bregma -3.36 mm

m/z 283-287 m/z 283-287

Grey Matter

White Matter

©2014 ImaBiotech

+ Diazepam Homogenate

(50 µg/g)

QC’s Sample preparation:

Frozen

Section

Mean 3 QC’s value

1 QC/image

1 QC/image

1 QC/image

Patent: WO 2012164221

Analytical variability: Inter-samples normalization

Replicate/Day 1 Replicate/Day 2 Replicate/Day 3

MSI

©2014 ImaBiotech

QC’s Control chart:

Patent: WO 2012164221

Analytical variability: Inter-samples normalization

QC’s Normalization Factor:

For inter-days/samples normalization, QCs…

Increase the reproducibility of MSI experiments

Provide better confidence into data interpretation.

QC = 1.02E+04

MS image n°

QCi (Int.) Coeff. = Qci / QC

1 1.09E+04 1.1

2 1.23E+04 1.2

3 7.52E+03 0.7

Normalization of MS images

©2014 ImaBiotech

Quantitation of Diazepam in Rat Brain using ILC

© ImaBiotech 2014

Small Molecule

Target molecules markers

Model IPF Rat Model (Bleomycin)

Sample Lung

Therapeutic area respiratory

Preparation Cryosection (10 µm)

Matrix DHB

ILC Matrix ions

Raster size 20 µm

Applications of QMSI

Instrumentation: MALDI-FTICR Mass Spectrometer Solarix (Bruker Daltonics) Matrix Automated Deposition Device SunCollect (SunChrom)

© ImaBiotech 2014

Readout Discovery: IPF Disease

Aim: identify some early readout markers of the IPF in a Bleomycin model

©2014 ImaBiotech

Readout Discovery: IPF Disease

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

Inte

nsi

ty

Fibrosis biomarkers intensity comparison between control (N=3) and IPF (N=6) tissues

Control

T7d

T22d

©2014 ImaBiotech

Conclusion

Analytical Variability = Quality Control (QC)

↑ Reproducibility/repeatability.

↓ Variation coeff.

Inter-samples comparison

↑ Confidence

Histological Variability = Internal standard (ILC)

Tissue Suppression Effect Normalization (Intra-)

See “real image”

ImaBiotech’s expertise: 200 QMSI projects

Success: 89% (limitation: sensitivity)

Average inter-sample variability: 15%-20% (based on the QCs)

Linearity: 2.5-3 Log

Best Limit of detection: 5ng/g

Average Limit of detection: 150ng/g

Quantinetix

Software

Multimaging Software

© ImaBiotech 2014

Dr. Jonathan Stauber Dr. David Bonnel Dr. Gregory Hamm Dr. Jean-Philippe Ebran Fabien Pamelard Alain Héron Raphaël Legouffe

Constance Buissart Guillaume Hochart Gael Picard-de-Muller Julien Stauber Emeline Falaux

Acknowledgement

Discovery Research Services, Edinburgh, United-Kingdom

Dr. Mary McELROY Dr. Stephen MADDEN

© ImaBiotech 2014

“A picture is worth a thousand words” Confucius (551–479 BC)

Thank you for your attention