IN VIVO METABOLISM OF [¹⁴C]RUBOXISTAURIN IN DOGS, MICE, AND RATS FOLLOWING ORAL ADMINISTRATION AND THE STRUCTURE DETERMINATION OF ITS METABOLITES BY LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY AND NMR SPECTROSCOPY

Abstract

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.

Pharmacokinetics, Metabolism, and Excretion of the Glycogen Synthase Kinase-3 Inhibitor LY2090314 in Rats, Dogs, and Humans: A Case Study in Rapid Clearance by Extensive Metabolism with Low Circulating Metabolite Exposure

“…LY2090314 is similar to ruboxistaurin in its clearance by extensive metabolism and predominant biliary/fecal elimination. Across mice, rats, dogs, and humans, 83-90% of the ruboxistaurin oral dose was recovered in feces, with #4% recovery in urine; in bile-cannulated rats, 59-66% of the oral dose was recovered in the bile as metabolites (Burkey et al, 2002;Barbuch et al, 2006). However, unlike LY2090314, ruboxistaurin metabolites collectively circulate at high systemic exposures, with the N-desmethyl metabolite circulating at 1.2-to 6.4-fold the parent exposure across species (Burkey et al, 2002;Barbuch et al, 2006).…”

“…Across mice, rats, dogs, and humans, 83-90% of the ruboxistaurin oral dose was recovered in feces, with #4% recovery in urine; in bile-cannulated rats, 59-66% of the oral dose was recovered in the bile as metabolites (Burkey et al, 2002;Barbuch et al, 2006). However, unlike LY2090314, ruboxistaurin metabolites collectively circulate at high systemic exposures, with the N-desmethyl metabolite circulating at 1.2-to 6.4-fold the parent exposure across species (Burkey et al, 2002;Barbuch et al, 2006). As a result of the structural differences, ruboxistaurin produces more N-dealkylation metabolites and exclusively undergoes secondary glucuronidation once nitrogens are exposed or oxygens are added by phase I metabolism Burkey et al, 2006).…”

“…Although several compounds have advanced to clinical testing in the maleimide kinase inhibitor class, including bisarylmaleimides ("BAMs," e.g., sotrastaurin) and bisindolylmaleimides ("BIMs," e.g., enzastaurin and ruboxistaurin), drug disposition and metabolism data are only available for ruboxistaurin in the literature (Burkey et al, 2002;Barbuch et al, 2006). LY2090314 is similar to ruboxistaurin in its clearance by extensive metabolism and predominant biliary/fecal elimination.…”

Inhibition of the adrenomedullin/nitric oxide signaling pathway in early diabetic retinopathy

“…During these 5 weeks, some animals were treated with the PKC β inhibitor RBX (30 mg/kg/day in chow). The dosage of RBX in the mouse chow provided by Eli Lilly for the in vivo experiments was chosen on the basis of their bioavailability and metabolic experiments [58].…”

Coupling of Liquid Chromatography/Tandem Mass Spectrometry and Liquid Chromatography/Solid-Phase Extraction/NMR Techniques for the Structural Identification of Metabolites following In Vitro Biotransformation of SUR1-Selective ATP-Sensitive Potassium Channel Openers

“…However, the SPE step (trapping and re-elution) has to be developed very carefully and could be sometimes the limitative and the time-consuming step of the process, especially with unknown products or very polar compounds (Wilson et al, 2006). Despite these disadvantages, LC/SPE/NMR, coupled with LC/tandem mass spectrometry (MS/MS) techniques, remains a convenient platform for accurate chemical structure elucidation (Barbuch et al, 2006;Yang, 2006;Tatsis et al, 2007). We decided to use such a platform for metabolism studies of SUR1-selective benzothiadiazine 1,1-dioxides.…”