title: Characterization of dark liver pigment observed in rats after subchronic dosing of the beta3-adrenergic receptor agonist LY368842.
abstract: Dark liver pigmentation was observed in F344 rats in a subchronic toxicology study after daily dosing of LY368842 glycolate. In addition, green-colored urine was observed in some animals. To identify the source of the pigment and its potential for toxic consequences, the liver pigment was isolated from the liver tissue of rats. The resulting material was a dark brown to black powder that was insoluble in water, organic solvents, or a tissue-solubilizing agent. Several techniques, such as chemical degradation, HPLC, tandem mass spectrometry (LC/MS/MS), (1)H NMR, and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), were employed to characterize the dark liver pigment. Following oxidative degradation of the isolated pigment, degradation products related to LY368842 were identified or tentatively identified using LC/MS/MS. Two degradation products had the same protonated molecular ion at m/z 505, which is 30 amu higher than that of LY368842. The major m/z 505 product has been identified as the indole-2,3-dione oxidative product based on (1)H NMR data and confirmed by an authentic standard. In addition, monohydroxylated product was also identified in the degradation mixture. These degradation products were consistent with the metabolites found in vivo in rats. MALDI-MS analyses of liver and urine pigment both identified a product with a protonated molecular ion at m/z 977, suggesting formation of indirubin-like and indigo-like pigments. The results obtained suggest that the oxidative metabolites of LY368842 played a key role in the formation of the liver and urine pigments.
authors.affiliation: Drug Disposition, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA. he_michelle_minxia@lilly.com
authors.forename: Minxia M
authors.initials: MM
authors.lastname: He
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Barbuch
authors.forename: Trent L
authors.initials: TL
authors.lastname: Abraham
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Lindsay
authors.forename: David A
authors.initials: DA
authors.lastname: Jackson
authors.forename: Bradley L
authors.initials: BL
authors.lastname: Ackermann
authors.forename: August V
authors.initials: AV
authors.lastname: Wilke
authors.forename: Charles B
authors.initials: CB
authors.lastname: Jensen
mesh_terms: D058667:Adrenergic beta-3 Receptor Agonists; D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000818:Animals; D007211:Indoles / Q000037:antagonists & inhibitors*; D007510:Isatin / Q000031:analogs & derivatives / Q000032:analysis; D008099:Liver / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010860:Pigments, Biological / Q000737:chemistry / Q000378:metabolism* / Q000652:urine; D011725:Pyridines / Q000037:antagonists & inhibitors*; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D058667:Adrenergic beta-3 Receptor Agonists; D000318:Adrenergic beta-Agonists; D007211:Indoles; C483012:LY368842; D010860:Pigments, Biological; D011725:Pyridines; D007510:Isatin