title: Using a simple HPLC approach to identify the enzymatic products of UTL-5g, a small molecule TNF-α inhibitor, from porcine esterase and from rabbit esterase.
abstract: UTL-5g is a novel small-molecule chemoprotector that lowers hepatotoxicity, nephrotoxicity, and myelotoxicity induced by cisplatin through TNF-α inhibition among other factors. As a prelude to investigating the metabolites of UTL-5g, we set out to identify the enzymatic products of UTL-5g under the treatment of both porcine liver esterase (PLE) and rabbit liver esterase (RLE). First, a number of mixtures made by UTL-5g and PLE were incubated at 25°C. At predetermined time points, individual samples were quenched by acetonitrile, vortexed, and centrifuged. The supernatants were then analyzed by reversed-phase HPLC (using a C18 column). The retention times and UV/vis spectra of individual peaks were compared to those of UTL-5g and its two postulated enzymatic products; thus the enzymatic products of UTL-5g were tentatively identified. Secondly, a different HPLC method (providing different retentions times) was used to cross-check and to confirm the identities of the two enzymatic products. Based on the observations, it was concluded that under the treatment of PLE, the major enzymatic products of UTL-5g were 5-methyliosxazole-3-carboxylic acid (ISOX) and 2,4-dichloroaniline (DCA). Treatment of UTL-5g by RLE also provided the same enzymatic products of UTL-5g from esterase. These results indicate that the peptide bond in UTL-5g was cleaved by PLE/RLE. Michaelis-Menten kinetics showed that the Km values of UTL-5g were 2.07mM with PLE and 0.37mM with RLE indicating that UTL-5g had a higher affinity with RLE. In summary, by a simple HPLC approach, we have concluded that the peptide bond in UTL-5g was cleaved by esterase from either porcine liver or rabbit liver in vitro and afforded DCA (at a mole ratio of 1:1) and ISOX. However, further studies are needed in order to determine whether UTL-5g is metabolized by microsomal enzymes to produce ISOX and DCA.
authors.affiliation: 21st Century Therapeutics, 1366 Hilton Road, Ferndale, MI 48220, USA.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Swartz
authors.forename: Yiguan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Frederick
authors.initials: F
authors.lastname: Valeriote
authors.forename: Ben
authors.initials: B
authors.lastname: Chen
authors.forename: Jiajiu
authors.initials: J
authors.lastname: Shaw
mesh_terms: D000814:Aniline Compounds / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004950:Esterases / Q000378:metabolism*; D007555:Isoxazoles / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D016014:Linear Models; D008958:Models, Molecular; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D013552:Swine; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors*
chemical_list: D000814:Aniline Compounds; D007555:Isoxazoles; D014409:Tumor Necrosis Factor-alpha; C559524:UTL-5g compound; C054619:2,4-dichloroaniline; D004950:Esterases
keywords: Enzymatic products; Esterase; HPLC approach; Metabolite; Michaelis–Menten kinetics; Peptide bond