1-P1-11 Utility of MALDI Imaging Mass Spectrometry in Drug Discovery: Histological Distribution of Chloroquine and Its Metabolite(s) in The Ocular Tissue of Pigmented Rats Hidefumi Kaji1, Yasuhiro Yamada1, Henry Y. Shion2, Motoji Oshikata3, and Yukari Haramaki3

1: DMPK Research Laboratory, Research Division, Mitsubishi Tanabe Pharma Corporation, Saitama, Japan, 2: Waters Corporation, Milford, MA, USA, 3: Nihon Waters, Tokyo, Japan

PURPOSE We previously reported that the histological distribution of chloroquine in

the retina, ocular melanin-containing tissue, of pigmented rats using matrix-assisted laser desorption/ionization (MALDI)-imaging quadrupole time-of-flight (Q-TOF) mass spectrometry (MS).Ref) The purpose of this study was to examine the histological distribution image of chloroquine and its metabolite(s) simultaneously obtained in this application in the retina of pigmented rats: the quantitative aspects and reproducibility were verified.

INTRODUCTION MALDI-imaging MS has emerged as a new technique for drug discovery and

development by several pharmaceutical companies. MALDI-imaging MS combines the benefits of both ARG and LC-MS/MS, and is capable of mapping the non-radiolabeled distribution of both the parent drug and its metabolites using visual-spatial images. This has allowed the relationship between efficacy or toxicity with drug concentration to be evaluated in greater detail and at lower cost than ARG and LC-MS/MS combined, thereby providing a powerful addition to the toolbox for the spatial analysis of tissue distribution in drug discovery.

METHODS At 24 h after single oral administration of chloroquine to pigmented rats, 10-µm sections taken from the single eyeball of each rat were prepared. The tissue sections were sprayed with MALDI Matrix CHCA using an automated sprayer, and analyzed using a Q-TOF MS equipped with MALDI source. The other eyeball was used for determining the concentrations of chloroquine by HPLC. The eyeball was solved in soluen-350 (Perkin Elmer), and then an aliquot of the sample was subjected to HPLC (Alliance 2695 HPLC system, Waters). MS condition for only chloroquine : Q m/z 320.18, product m/z 247.1 was used for imaging MS condition for both chloroquine and n-desethylchloroquine : Q m/z 320.18, product m/z 142.15 was used for imaging Q m/z 292.18, product m/z 114.12 was used for imaging

MALDI Imaging MS Process for Rat Eyeball Sections

R2 = 0.9802

0

2000

4000

6000

8000

10000

12000

0 5 10 15 20

HPLC (µg/g tissue)

MA

LDI (

inte

nsity

/slic

e)

Mean±S.D., n=6-12

Correlation of MALDI and HPLC Data for Chloroquine in Rat Eyeball

The signal intensities of the major fragment ion, m/z 247, of chloroquine in plasma (with or without melanin) images corresponded well with the chloroquine concentrations.

Group Intensity* Intra-CV% Intensity Inter-CV%

Vehicle Cont. 55 ± 5 9.1

2 mg/kg, 1st 216 ± 56 26.4

2 mg/kg, 2nd 887 ± 156 17.6

2 mg/kg, 3rd 1373 ± 332 24.2 901 ± 520 57.8

6 mg/kg, 1st 2480 ± 46 1.8

6 mg/kg, 2nd 3957 ± 997 24.9 3218 ± 1022 31.8

20 mg/kg, 1st 8917 ± 444 5.0

20 mg/kg, 2nd 6703 ± 902 13.5

20 mg/kg, 3rd 8643 ± 1313 15.2

20 mg/kg, 4th 10367 ± 404 3.9 8658 ± 1543 17.8

*: Each value represents the mean ± S.D. of triplicate determinations.

Chloroquine (CQ)

N-desethylchloroquine (DCQ)

Bisdesethylchloroquine (BDCQ)

320

292

264

MS spectrum Product ion

320

247

164

142

292 247

179 114

264 247

179

142

247 179

114

86

Localizing Chloroquine and Its Metabolite in Pigmented Rat Eyeball

•Chloroquine and one of its metabolites, n-desethylchloroquine, were detected in the MS/MS mode by monitoring of their specific fragment ions, m/z 142 and 114, respectively, and imaged through the rat eyeball.

•The distribution of the metabolite was similar to that observed for chloroquine in terms of the histological images and specific localization to retina.

[m/z 142] [m/z 114] [CQ 20 mg/kg, po]

[Vehicle control]

CONCLUSIONS Chloroquine and n-desethylchloroquine were simultaneously analyzed by

MALDI imaging MS: the quantitative aspects and reproducibility of the specific localization of chloroquine were verified.

The distribution of the metabolite was similar to that observed for chloroquine in terms of the histological images and specific localization to retina.

This technology can provide relevant information concerning the molecular mechanisms underlying drug efficacy and/or safety in drug discovery.

RESULTS

Localizing Chloroquine in Pigmented Rat Eyeball Plate 1 Plate 2 Plate 3 Plate 1 Plate 2 Plate 3

Plate 1 Plate 2 Plate 3 Plate 1 Plate 2 Plate 3

[Vehicle control] [CQ 2 mg/kg, po] 2.1 µg/g tissue by HPLC

[CQ 6 mg/kg, po] [CQ 20 mg/kg, po] 18.5 µg/g tissue by HPLC 4.8 µg/g tissue by HPLC

Photo

MALDI Image (Contrast: 0-15,000)

Ref.) Yamada Y. et al., Rapid Commun. Mass Spectrom., 25: 1600-1608 (2011).

Embedding in OCT Compound, Cryosectioned (10 µm)

Mounted onto standard microscope glass plate

Eyeball isolation at 24 h

Brown Norway Rat

Chloroquine 2, 6, or 20 mg/kg, po

Mounted onto MALDI plate

MALDI IMS

Mass (m/z) specific ion images using

BioMap

Matrix (CHCA*) Sprayed by

TM-Sprayer™

*: CHCA (α-cyano-4-hydroxycinnamic acid)

Eyeball Harderian gland

Four groups: 1) control & 20 mg/kg, 2) 2 mg/kg & 20 mg/kg 3) 6 mg/kg & 20 mg/kg, 4) 2 mg/kg & 6 mg/kg

Quantitative Aspects of Chloroquine in Rat Plasma by MALDI Imaging MS

The signal intensities of the major fragment ion, m/z 247, of chloroquine within the retina in distribution images corresponded well with the chloroquine concentrations in ocular tissues measured by HPLC.

0.3 µg/mL

3.0 µg/mL

30 µg/mL

10 µg/mL

1.0 µg/mL

0.1 µg/mL Blank

[MALDI Image] [Spot of Plasma Sample] [Examples of imaging areas] 5 wells included for each concentration

[H&E staining]

Pupil

Iris Retina Harderian gland

Crystalline lens (Lens should be present in the area within dotted line)

Cornea

(R2= 0.9979)

0

5.0E+06

1.0E+07

1.5E+07

2.0E+07

2.5E+07

3.0E+07

0 10 20 30 Concentration (µg/mL)

Sign

al In

tens

ity with melanin

without melanin (R2= 0.9932)

dos Santos Magalhães I.R. and Sueli Bonato P., J. Sep. Sci., 31: 3109 (2008)

Application of Ion Mobility (IM) Separation for MALDI Imaging MS

Ion mobility separation effectively reduced the background signals.

Without IM separation

With IM separation

[Vehicle control] [CQ 2 mg/kg, po]

a)

c)

b)

d)

a)

c)

b)

d)

[Mass Spectrum]