Abstract

Ruboxistaurin (LY333531), a potent and isoform-selective protein kinase C β inhibitor, is currently undergoing clinical trials as a therapeutic agent for the treatment of diabetic microvascular complications. The present study describes the disposition and metabolism of [¹⁴C]ruboxistaurin following administration of an oral dose to dogs, mice, and rats. The study revealed that ruboxistaurin was highly metabolized in all species. Furthermore, the results from the bile duct-cannulated study revealed that ruboxistaurin was well absorbed in rats. The primary route of excretion of ruboxistaurin and its metabolites was through feces in all species. The major metabolite detected consistently in all matrices for all species was the N-desmethyl metabolite 1, with the exception of rat bile, in which hydroxy N-desmethyl metabolite 5 was detected as the major metabolite. Other significant metabolites detected in dog plasma were 2, 3, 5, and 6 and in mouse plasma 2, 5, and 19. The structures of the metabolites were proposed by tandem mass spectrometry with the exception of 1, 2, 3, 5, and 6, which were additionally confirmed either by direct comparison with authentic standards or by nuclear magnetic resonance spectroscopy. To assist identification by nuclear magnetic resonance spectroscopy, metabolites 3 and 5 were produced via biotransformation using recombinant human CYP2D6 and, likewise, metabolite 6 and compound 4 (regioisomer of 3 which did not correlate to metabolites found in vivo) were produced using a microbe, Mortierella zonata. The unambiguous identification of metabolites enabled the proposal of clear metabolic pathways of ruboxistaurin in dogs, mice, and rats.