In Situ Mass Spectrometry Imaging and Ex Vivo Characterization of Renal Crystalline Deposits Induced in Multiple Preclinical Drug Toxicology Studies

Drug toxicity observed in animal studies during drug development accounts for the discontinuation of many drug candidates, with the kidney being a major site of tissue damage. Extensive investigations are often required to reveal the mechanisms underlying such toxicological events and in the case of crystalline deposits the chemical composition can be problematic to determine. In the present study, we have used mass spectrometry imaging combined with a set of advanced analytical techniques to characterize such crystalline deposits in situ. Two potential microsomal prostaglandin E synthase 1 inhibitors, with similar chemical structure, were administered to rats over a seven day period. This resulted in kidney damage with marked tubular degeneration/regeneration and crystal deposits within the tissue that was detected by histopathology. Results from direct tissue section analysis by matrix-assisted laser desorption ionization mass spectrometry imaging were combined with data obtained following manual crystal dissection analyzed by liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy. The chemical composition of the crystal deposits was successfully identified as a common metabolite, bisulphonamide, of the two drug candidates. In addition, an un-targeted analysis revealed molecular changes in the kidney that were specifically associated with the area of the tissue defined as pathologically damaged. In the presented study, we show the usefulness of combining mass spectrometry imaging with an array of powerful analytical tools to solve complex toxicological problems occurring during drug development.De två första författarna delar förstaförfattarskapet.De två sista författarna delar sistaförfattarskapet.</p

Results from an untargeted analysis of kidney tissue from drug treated animals and vehicle controls.

<p>A) Total ion chromatogram of kidney extract from the LC-MS analysis of vehicle control (upper) and animal dosed with compound 1 (low crystal load) (lower), run on an HILIC column. B) Mass spectrum from the peak marked in red on panel A, showing a PEG distribution with <i>m/z</i> 44 distance between peaks. C) The distribution of PEG on the tissue is represented by the ion distribution image of <i>m/z</i> 437. This distribution pattern is overlaid on the tissue sections from (i) vehicle control, (ii) animal dosed with compound 1 (low crystal load), (iii) animal dosed with compound 1 (high crystal load), and (iv) animal dosed with compound 2 (not formulated in PEG400). The MSI samples were coated with CHCA and analyzed in positive mode on the UltraFlex II. The data was normalized by total ion count.</p

The structures of the two administered compounds (compound 1; 4-(phenylethynyl)-N-[(2-sulfamoylphenyl)sulfonyl]benzamide, compound 2; 4-(cyclobutylethynyl)-N-[(2-sulfamoylphenyl)sulfonyl] benzamide), and their common metabolite bisulphonamide (benzene-1,2-disulphonamide).

<p>The structures of the two administered compounds (compound 1; 4-(phenylethynyl)-N-[(2-sulfamoylphenyl)sulfonyl]benzamide, compound 2; 4-(cyclobutylethynyl)-N-[(2-sulfamoylphenyl)sulfonyl] benzamide), and their common metabolite bisulphonamide (benzene-1,2-disulphonamide).</p

Identification and mapping of renal crystalline deposits.

<p>A) i; Mass spectrum of crystals isolated from kidney tissue analyzed by LC-MS. Retention time and mass-to-charge ratio matches that of bisulphonamide standard. ii; MS/MS spectrum of 234.98 observed in the crystal isolate. The fragmentation pattern matches that of bisulphonamide standard. B)¹H-NMR spectra of the aromatic proton region of crystals from kidney tissue dissolved in DMSO. C) Optical images of analyzed tissue sections. i) vehicle control, ii) bisulphonamide standard on vehicle control, iii) compound 1 dosed tissue with a low crystal load, iv) compound 1 dosed tissue with a high density of crystals, v) compound 2 dosed tissue with a high crystal load. All samples were coated with sinapinic acid and analyzed in negative mode on the G2 Synapt. The data was normalized by total ion count. D) Ion distribution of bisulphonamide (m/z 235) on the tissue sections in panel C. The color intensity scale is adjusted to 2% of the maximum intensity on tissue v in order to visualize the distribution patterns on all tissues using the same color intensity scale. This means that pixels on the tissues in panel D above this value appear saturated. Data was acquired at a spatial resolution of 100 µm. E) Ion distribution of bisulphonamide overlaid on the tissue sections from the animal with a low crystal load following administration of compound 1. Left; ion distribution image of <i>m/z</i> 235 overlaid on scanned image of tissue section. Crystals are marked with arrows. Right; scanned image following H&E staining of the same tissue with example of a crystalline deposit in the kidney surrounded by a slight mononuclear cell reaction. Crystals are marked with arrows and circled in green. The size of the majority of the crystals ranged between 50 and 100 µm.</p

Ion distribution images of administered drug candidate compounds and some detected metabolites.

<p>A) Optical images of analyzed tissue sections. i; vehicle control, ii; compound 1 dosed animal tissue section with low crystal load, iii; compound 1 dosed animal tissue section with high density of crystals. B) Ion distribution of compound 1 (<i>m/z</i> 439) on the tissue sections in panel A. C) Optical images of analyzed tissue sections. i; vehicle control, ii; compound 2 dosed animal tissue section. D) Ion distribution image of compound 2 on the tissue sections in panel C. E) Ion distribution image of a compound 2 metabolite (doubly oxidized) on the tissue sections in panel C. F) Ion distribution image of compound 2 metabolite (triply oxidized) on the tissue sections in panel C. The samples were coated with sinapinic acid and analyzed in negative mode on the Synapt G2. The data was normalized by total ion count.</p