Guillaume Hochart , MS Imaging Department, Imabiotech, Loos, France
Joseph Marini , Covance Laboratories Inc, Madison, WI
Aurore Tomezyk , Imabiotech, Loos, France
Phil Mantefeul , Covance Laboratories Inc, Madison, WI
Bonnie Jung , Covance Laboratories Inc, Madison, WI
David Bonnel , Imabiotech, Loos, France
Stefan Linehan , Imabiotech, Loos, France
Don Mckenzie , Covance Laboratories Inc, Madison, WI
Jonathan Stauber , Imabiotech, Loos, France
Mass Spectrometry Imaging (MSI), known to bring molecular distribution information in a specific mass range, allows the quantification of compounds in biological tissues (QMSI). This function has been well established with Quantitative Whole Body Autoradiography (QWBA), the gold standard technique in preclinical imaging. However, QWBA lacks the ability to differentiate between drug components (e.g. parent and metabolites) and establish separate quantitative results for each target tissue of interest. This study demonstrates the QMSI capabilities as a complementary approach to QWBA to follow chloroquine and one of its metabolites (desethyl-chloroquine) at different time-points after a single administration of a radiolabeled dose to Long-Evans male rats (30 mg/kg). Rats were euthanized under deep anesthesia (isoflurane) at 4, 24, 72, 168, and 336 hours post-dose. Carcasses were then immediately frozen in a hexane/dry ice bath for approximately 8 minutes, embedded in chilled carboxymethylcellulose and frozen into blocks for analysis. Sections were collected onto adhesive tape for QWBA and QMSI analysis in the sagittal plane. QWBA images were obtained following exposure to phosphorimaging screens and scanned using a GE Typhoon scanner. QWBA images were generated and quantified against 14C fortified standards using MCIDTM Analysis software [InterFocus Imaging LTS.]. For QMSI, MALDI matrix spiked with labeled chloroquine-d4 and its metabolite desethyl chloroquine-d4 were sprayed onto the slide. Analyses were performed by a 7T MALDI-FTICR in the head region of the animal targeting the eye and in the mid whole-body region for other organs of interest. Distribution and quantification were performed with Quantinetix software using ILC (Isotopic Labeled Compound) approach. 1H-Chloroquine and 1H-desethyl chloroquine were well detected by MALDI-FTICR in the eye including; the uveal tract, the vitreous humor, the lens and the Harderian gland. In the mid whole-body region, both compounds were also detected in various organs from the unique prepared T4h sections. Interestingly the two compounds were co-localized into the tissue sections and their distributions matched the zones obtained by QWBA. In QWBA additional regions containing the radiolabeled moiety could be clearly identified because of the high sensitivity of the technique. The advantage brought by MALDI QMSI was the ability to discriminate between parent drug 1H-choloroquine and its metabolite 1H-desethyl chloroquine so that each compound had its own distribution image and thus its own quantification data directly in one tissue section. The used labeled forms of both compounds during the matrix deposit allowed normalizing the data for each position targeted with the MALDI onto the section of interest and the calibration range of both 1H-choloroquine and 1H-desethyl chloroquine and quantifying each compound into the organs of interest with the ILC approach. Finally the QMSI was able to demonstrate the disappearance of the drug and its metabolite with time to better understand differential pharmacokinetic analysis demonstrating the additional input of the technology compared to QWBA.