1. Studies were conducted in the Fischer 344 rat and beagle dog to determine the disposition of LY333531 and its equipotent active des-methyl metabolite, LY338522, both potent and selective inhibitors of the beta-isozyme of protein kinase C. 2. Male Fischer 344 rats and female beagle dogs received a single 5-mg kg(-1) oral dose of (14)C-LY333531. Urine, faeces, bile and plasma were collected and analysed for (14)C, LY333531 and LY338522. 3. LY333531 was eliminated primarily in the faeces (91% by 120 h in rat, 90% by 96 h in dog). Bile contributed the majority of the radioactivity excreted in the faeces in rat (66% in the cannulated bile duct study) and a variable but significant proportion in dog. 4. Pharmacokinetics following a single 5 mg kg(-1) oral dose of (14)C-LY333531 to the male rat produced C(max) and AUC(0-infinity ) for LY333531 of 14.7 ng ml(-1) and 60.8 ng h ml(-1), respectively, with a half-life of 2.5 h. LY338522 and total radioactivity showed similar profiles. 5. In the female dog at the same dose, C(max) and AUC(0-infinity ) of LY333531 were higher, producing 245 +/- 94 ng ml(-1) and 1419 +/- 463 ng h ml(-1), respectively, with a half-life of 5.7 h. 6. The data indicate that the disposition of LY333531 is similar in rat and dog.
ABSTRACT:Ruboxistaurin (LY333531), a potent and isoform-selective protein kinase C  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 [ 14 C]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.
ABSTRACT:Ruboxistaurin is a potent and specific inhibitor of the  isoforms of protein kinase C (PKC) that is being developed for the treatment of diabetic microvascular complications. The disposition of [ 14 C]ruboxistaurin was determined in six healthy male subjects who received a single oral dose of 64 mg of [ 14 C]ruboxistaurin in solution. There were no clinically significant adverse events during the study. Whole blood, urine, and feces were collected at frequent intervals after dosing. Metabolites were profiled by high performance liquid chromatography with radiometric detection. The total mean recovery of the radioactive dose was approximately 87%, with the majority of the radioactivity (82.6 ؎ 1.1%) recovered in the feces. Urine was a minor pathway of elimination (4.1 ؎ 0.3%). The major route of ruboxistaurin metabolism was to the N-desmethyl ruboxistaurin metabolite (LY338522), which has been shown to be active and equipotent to ruboxistaurin in the inhibition of PKC  . In addition, multiple hydroxylated metabolites were identified by liquid chromatography-mass spectrometry in all matrices. Pharmacokinetics were conducted for both ruboxistaurin and LY338522 (N-desmethyl ruboxistaurin, 1). These moieties together accounted for approximately 52% of the radiocarbon measured in the plasma. The excreted radioactivity was profiled using radiochromatography, and approximately 31% was structurally characterized as ruboxistaurin or N-desmethyl ruboxistaurin. These data demonstrate that ruboxistaurin is metabolized primarily to N-desmethyl ruboxistaurin (1) and multiple other oxidation products, and is excreted primarily in the feces.Protein kinase C (PKC) is a group of isozymes important in signal transduction and intracellular signaling through diacylglycerol. In diabetes, hyperglycemia-induced generation of diacylglycerol selectively activates the  2 isoform of PKC, and increases in diacylglycerol and PKC activity have been measured in the retina, kidneys, aorta, and heart, all organs that are known to develop diabetic complications associated with the vasculature (Craven and DeRubertis, 1989;Craven et al., 1990;Ayo et al., 1991;Inoguchi et al., 1992; Schrier, 1992, 1993;Shiba et al., 1993;DeRubertis and Craven, 1994;Kikkawa et al., 1994). The selective inhibition of PKC  has been shown to inhibit diabetes-induced abnormalities in retinal blood flow and intraocular neovascularization caused by retinal ischemia in animals (Danis et al., 1998;Kowluru et al., 1998). These data suggest that hyperglycemia and the resulting activation of PKC  may contribute to the development of diabetic complications. Furthermore, a selective PKC  inhibitor may be of benefit in the treatment of diabetic complications by blocking this mechanism.Ruboxistaurin ( Fig. 1) . Previous studies have shown that ruboxistaurin (LY333531) was effective in reversing vascular abnormalities induced in diabetic animals (Ishii et al., 1996) and that diabetes-induced elevations in PKC activity in the retina and kidneys of diabetic animals wer...
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