Qualitative & Quantitative MS imaging technique for BAK distribution in eye. Stauber Jonathan, PhD CSO ImaBiotech

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Maurice Barton
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1 Q Qualitative & Quantitative MS imaging technique for BAK distribution in eye Stauber Jonathan, PhD CSO ImaBiotech EBF congress 2012, Bruxelles

2 Q An innovative company with an innovative technology WHAT & WHY?

Technology in brief Compound administration and biopsy (in Drug discovery studies) Histological sectioning of specific organ or Whole Body Q Advantages: Do not need labeling More information : Drug +

3 Technology in brief Compound administration and biopsy (in Drug discovery studies) Histological sectioning of specific organ or Whole Body Q Advantages: Do not need labeling More information : Drug + metabolites distribution Short study : 2-4 weeks H&E and IHC Slide preparation: matrix with best output chosen for the sample; automated method for matrix deposition developed by our experts Matrix-assisted Laser Desorption/Ionization (MALDI) mass spectrometry. Spectra acquired from MALDI data using variety of modes MS, MS/MS, SRM/MRM Images constructed from correlated spectra using individual colors for each molecule easy discrimination of molecules in a single experiment 3

4 Where do you find MALDI imaging? Target identification HTS Hit-to-Lead Lead Generation Lead Optimization Pre-candidate Candidate Preclinical Development Phase I III NDA FDA review Approval Q In vivo Efficacy Safety Assessment DMPK FIH Dose Prediction Biomarkers IND Filing Pre lead compounds validation Assess lead distribution Assess Toxicity Combination with QWBA 4

5 Where do you find MALDI imaging? Target identification HTS Hit-to-Lead Lead Generation Lead Optimization Pre-candidate Candidate Preclinical Development Phase I III NDA FDA review Approval Q In vivo Efficacy Safety Assessment DMPK FIH Dose Prediction Biomarkers IND Filing Pre Lead compounds evaluation 30 images / 5 Drugs Time points: 1 Time: 1 month Assess lead distribution with Quantitation # Images: 20 (20-500µm) Time points: 5 Time: 1 month Assess Toxicity and quantitation # Images: 20 drug+metabolites Time points: 5 Time: 1 month Combination with QWBA # Images: 20 Time points: 5 Time: 1 month 5

6 Q Taking the next step QUANTITATIVE MS IMAGING

Technology in brief Compound administration and biopsy (in Drug discovery studies) Histological sectioning of specific organ or Whole Body Q Drawback : Ion suppression ontissue No internal standard

7 Technology in brief Compound administration and biopsy (in Drug discovery studies) Histological sectioning of specific organ or Whole Body Q Drawback : Ion suppression ontissue No internal standard H&E and IHC Slide preparation: matrix with best output chosen for the sample; automated method for matrix deposition developed by our experts Matrix-assisted Laser Desorption/Ionization (MALDI) mass spectrometry. Spectra acquired from MALDI data using variety of modes MS, MS/MS, SRM/MRM Consequence : No quantitative Images constructed from correlated spectra using individual colors for each molecule easy discrimination of molecules in a single experiment 7

8 Quantitation by MALDI Imaging What the matrix effect? Optical Image Matrix Standard TEC = Int Tissue Int Plate Eye Lung Stomach Spleen Kidney Q 1,00 Examples of mouse whole body TEC values Thym us Heart Liver BLadder 0,80 0,60 0,40 0,20 0,00 Distribution de m/z «S42305» MALDI Image 8

Quantitation by MALDI Imaging Two ways of quantitation by MS imaging : 1. Labeled technique 2. No Labeled technique Matrix deposition with Drug standard 1.

9 Quantitation by MALDI Imaging Two ways of quantitation by MS imaging : 1. Labeled technique 2. No Labeled technique Matrix deposition with Drug standard 1. Each position normalized by pseudo internal standard Dosed Animal Q Whole body section Matrix Labeled Standard Matrix deposition with Drug standard 2. Each position is normalized by TEC values from each region of interest Control Whole body section Matrix Standard Dosed Animal Whole body section Matrix 9

10 Example of Olanzapine Quantitation Software : Quantinetix Q 10

11 Example of Olanzapine Quantitation Q 11

12 Conc. (µg/g tissue) Example of Olanzapine Quantitation Olanzapine dosage comparison q vs LC-MS 2 Q 50,0 45,0 40,0 35,0 30,0 25,0 20,0 15,0 10,0 5,0 0,0 q LC-MS2 µg/g tissue 37,3 41,1 12

13 Concentration (µg/g tissue) Concentration (µg/g tissue) Quantitation by MS imaging (q) Propranolol q vs qwba * BDM31343 q vs LCMS² Q 10 WBA q 20 LC MS/MS q 5 0 Kidney Lung Brain 10 0 Lung Tissue qwba (µg/g tissue) q (µg/g tissue) CV% Kidney Lung Tissue LCMS² (µg/g tissue) q (µg/g tissue) CV% Lung Brain * Kertesz et al, Analytical Chemistry (13),

14 Q COMBINED IMAGING TECHNIQUES TO COMPOUND DISTRIBUTION & TOX STUDIES

Ocular drug distribution Benzalkonium (BAK) containing antiglaucoma eye drops have been reported to cause ocular surface disorders with tear film alteration, eye irritation and to promote dry eye.

15 Ocular drug distribution Benzalkonium (BAK) containing antiglaucoma eye drops have been reported to cause ocular surface disorders with tear film alteration, eye irritation and to promote dry eye. BAK was also suspected to induce cystoid macular edema following cataract surgery in BAK-receiving eyes. use new mass spectrometry imaging () techniques to describe the BAK distribution in eye in an in vivo model and investigate the physiopathological consequences at the molecular level. Low Chronic model (LCm): twice a day for 5 months, 1 drop of 0.01% BAK the commonly concentration used in eye drops High Sub Chronic model (HSCm): 1 drop of 0.2% BAK once a day for 1 month. MALDI Imaging / H&E / Immunohistochemistry Q 15

High resolution ocular drug distribution

609 R: 2274 m/z 304 m/z 304 m/z 212 Q 100%

32) myristalkonium C14 (C23H42N+, m/z 332.

outer periphery of eyeball, in the cornea

16 High resolution ocular drug distribution BAK C12 Distribution in rabbit eye Trabeculum (a) Limbus (b) (c) Sclerotic-cornea junction Trabeculum S/N: 609 R: 2274 m/z 304 m/z 304 m/z 212 Q 100% 100% 500 µm 500 µm 500 µm 0% 0% 1 mm H.E. staining benzododecinium C12 (C21H38N+, m/z ) myristalkonium C14 (C23H42N+, m/z ). ion showed a distribution in the outer periphery of eyeball, in the cornea and conjunctiva as well as in the limbus and near the iridocorneal angle and the trabecular meshwork. 16

10µm imaging of BAK H&E staining of eye section at 1 month BAK C 12 m/z 304.

17 10µm imaging of BAK H&E staining of eye section at 1 month BAK C 12 m/z BAK C 14 m/z Q Cornea BAK C 12 BAK C 14 spatial resolution: 30 µm 17

18 10µm imaging of BAK H&E staining of eye section at 1 month BAK C 12 m/z BAK C 14 m/z Q Iridocorneal angle BAK C 12 BAK C 14 18

19 10µm imaging of BAK H&E staining of eye section at 1 month BAK C 12 m/z BAK C 14 m/z Q Retina/Choroid/Sclera BAK C 12 BAK C 14 19

matrix effect) 1,00 0,90 0,80 0,70 0,60 0,50 0,40 0,30 0,20 BAK C 12 C12 0,33

20 TEC Values Quantitation of BAK by MS Imaging H&E staining of control eye section BAK C 12 BAK C 14 T Q S BAK C 12 BAK C 14 BAK C 12 TEC calculation (Biological matrix effect) 1,00 0,90 0,80 0,70 0,60 0,50 0,40 0,30 0,20 BAK C 12 C12 0,33 0,30 0,23 0,19 0,36 0,32 0,56 0,50 0,83 0,74 0,53 0,54 0,54 0,50 0,60 0,55 0,10 0,00 20

21 TEC Values Quantitation of BAK by MS Imaging H&E staining of control eye section BAK C 12 BAK C 14 T Q S BAK C 12 BAK C 14 BAK C 12 TEC calculation (Biological matrix effect) 1,00 0,90 0,80 0,70 0,60 0,50 0,40 0,30 0,20 BAK C 12 C12 0,33 0,30 0,23 0,19 0,36 0,32 0,56 0,50 0,83 0,74 0,53 0,54 0,54 0,50 0,60 0,55 0,10 0,00 21

Region Quantitation of BAK by MS Imaging BAK C 12 BAK C 14 Q TEC calculation (Biological matrix effect) Compound BAK C12 (µg/g of tissue) BAK C 14 (µg/g of tissue) LOQ (µg/g of tissue)

22 Region Quantitation of BAK by MS Imaging BAK C 12 BAK C 14 Q TEC calculation (Biological matrix effect) Compound BAK C12 (µg/g of tissue) BAK C 14 (µg/g of tissue) LOQ (µg/g of tissue) Sample/Replicate N 1 N 2 N 1 N 2 BAK C 12 BAK C 14 Sclera b.l.q b.l.q b.l.q b.l.q Choroid b.l.q b.l.q b.l.q b.l.q Retina b.l.q b.l.q b.l.q b.l.q Fibreous b.l.q b.l.q b.l.q b.l.q Aqueous Humor b.l.q Iridocorneal Angle b.l.q b.l.q b.l.q b.l.q Cornea

23 Toxicity Study The number of CD45 positive cells increased in all areas of interest and were higher in the HSCm in cornea, conjunctiva, limbus, trabecular meshwork. Vimentin expression increased in in all retinal layers in treated eyes suggesting a microglial cell activation. Q 23

24 BAK distribution and study Ion images and immunohistology were put side by side to correlate inflammatory areas. MALDI-ToF/ToF imaging confirmed these data and also showed the presence of BAK in the retina and near the optic nerve. All these localizations were confirmed with the BAK 0.2% model. The number of CD45 positive cells increased in all areas of interest and were higher in the HSCm in cornea, conjunctiva, limbus, trabecular meshwork. Vimentin expression increased in in all retinal layers in treated eyes suggesting a microglial cell activation. imaging a technique of interest = Especially in small organs (difficult to dissect) or for Whole body distribution with 25 organs to study Q Useful with the combination of H&E + Immuno Assay Quantitative MS imaging approaches have been presented with some limitation of pseudo internal standards and the limitation of labeled compound 24

25 Dr. Grégory Hamm Dr. David Bonnel Fabien Pamelard Raphaël Legouffe Dr. Jonathan Stauber Pr. Françoise Brignole-Baudouin Dr. Nicolas Desbenoit Pr. Christophe Baudouin Hong Liang Razika Nanache Grant ANR PIRIbio MASDA-EYE Dr. Jean-Pierre Both Pr. Alain Brunelle Pr. Isabelle Fournier Dr. Vincent Guérineau Dr. David Touboul Dr. Maxence Wisztorski Pr. Olivier Laprévote Q 25

Keeping an Eye on Molecular Imaging: Drug Efficacy & Toxicity in Ophthalmology

Application Note #MSI-11 Keeping an Eye on Molecular Imaging: Drug Efficacy & Toxicity in Ophthalmology Introduction Mass spectrometry Imaging (MSI) applications for ophthalmic drug discovery have recently