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 Kaji 1 , Yasuhiro Yamada 1 , Henry Y. Shion 2 , Motoji Oshikata 3 , and Yukari Haramaki 3 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 R 2 = 0.9802 0 2000 4000 6000 8000 10000 12000 0 5 10 15 20 HPLC (μg/g tissue) MALDI (intensity/slice) Mean± S.D., n=6-12 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, 1 st 216 ± 56 26.4 2 mg/kg, 2 nd 887 ± 156 17.6 2 mg/kg, 3 rd 1373 ± 332 24.2 901 ± 520 57.8 6 mg/kg, 1 st 2480 ± 46 1.8 6 mg/kg, 2 nd 3957 ± 997 24.9 3218 ± 1022 31.8 20 mg/kg, 1 st 8917 ± 444 5.0 20 mg/kg, 2 nd 6703 ± 902 13.5 20 mg/kg, 3 rd 8643 ± 1313 15.2 20 mg/kg, 4 th 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 • 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 *: 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 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 (R 2 = 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) Signal Intensity with melanin without melanin (R 2 = 0.9932) dos Santos Magalhães I.R. and Sueli Bonato P., J. Sep. Sci., 31: 3109 (2008) 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]