Metabolic activation of a pentafluorophenylethylamine derivative: formation of glutathione conjugates in vitro in the rat.

The aim was to investigate the metabolic activation potential of a pentafluorophenylethylamine derivative (compound I) in vitro in the rat and to identify the cytochrome P450 (CYP) enzymes that catalyse these metabolic activation processes. Reduced glutathione (GSH) was fortified in rat hepatocytes and liver microsomes to trap possible reactive intermediates. Four glutathione conjugates (M1-4) were identified by LC-MS(n) following incubation of compound I in GSH-enriched rat hepatocytes and liver microsomes. Three of these conjugates (M2-4) have not been reported previously for pentafluorophenyl derivatives. Elemental composition analysis of these conjugates was obtained using high-resolution quadrupole time-of-flight mass spectrometry. The formation of GSH conjugate M1 was rationalized as a direct nucleophilic replacement of fluoride by glutathione, whereas the formation of the GSH conjugates M2-4 was proposed to occur by NADPH-dependent metabolic activation of the pentafluorophenyl ring via arene oxide, quinone and/or quinoneimine reactive intermediates. Formation of these conjugates was enhanced three- to five-fold in liver microsomes obtained from phenobarbital- and dexamethasone-treated rats. In incubations with pooled rat liver microsomes and recombinant rat CYP3A1 and CYP3A2, troleandomycin (TAO) reduced the formation of GSH conjugates M2-4 by 80-90%, but it had no effect on the formation of M1. Incubation of compound I with rat supersomes indicated that only CYP3A1 and CYP3A2 were capable of mediating these metabolic activation processes.

A semi-automated method for measuring the potential for protein covalent binding in drug discovery.

INTRODUCTION: Covalent protein binding of metabolically reactive intermediates of drugs has been implicated in drug toxicity including the occurrence of idiosyncratic drug toxicity. Investigators therefore would prefer to avoid developing compounds that produce significant amounts of reactive metabolites. By incubating the radiolabeled drug of interest with liver microsomes it is possible to evaluate the propensity of a drug candidate to covalently bind to proteins. METHODS: Here we present a semi-automated method in which a Brandel cell harvester is used to collect and wash proteins that have been incubated with radiolabeled drug. This method utilizes glass fiber filter paper to capture precipitated protein, rather than the more traditional exhaustive extraction/centrifugation approach. Using model compounds (including [14C]diclofenac, [3H]imipramine, [14C]naphthalene, and [14C]L-746530) we compare the covalent binding results obtained using this method to results generated using the traditional method and we performed cross-laboratory testing of assay reproducibility. RESULTS: It was found that results from new method correlated highly with the traditional method (R2=0.89). The cross-laboratory testing of the method showed an average interlaboratory coefficient of variation of only 18.4%. DISCUSSION: This method provides comparable results to the more traditional centrifugation-based method with considerable time and labor savings.

Use of on-line hydrogen/deuterium exchange to facilitate metabolite identification.

Biotransformation studies performed on an investigational compound (I, represented by R1-CH(NH(2))-CO-N(R2)-CH(2)-S-R3) led to the identification of five metabolites (M1-M5). Based on LC/MS (liquid chromatography/mass spectrometry) analysis which included the use of H(2)O and D(2)O in the mobile phases, they were identified as the sulfoxide (M1), sulfone (M2), carbamoyl glucuronide (M3), N-glucuronide (M4), and N-glucoside (M5) metabolites, respectively. The structure of M3, a less commonly seen carbamoyl glucuronide metabolite, was established using on-line H/D (hydrogen/deuterium) exchange experiments conducted by LC/MS. H/D exchange experiments were also used to distinguish the S-oxidation structures of M1 and M2 from hydroxylation. Herein, the application of deuterium oxide as the LC/MS mobile phase for structural elucidation of drug metabolites in biological matrices is demonstrated.

Growth and subsequent feedlot performance of estradiol-implanted vs nonimplanted steers grazing fall-accumulated endophyte-infested or low-endophyte tall fescue.

A growing-finishing study using Angus steer calves was conducted in three phases: 1) grazing stockpiled 'Kentucky-31' tall fescue (Festuca arundinacea Schreb.) with high (65%; HE KY-31) and low (0%; LE KY-31) infestation rates of Acremonium coenophialum Morgan-Jones and Gams and 'Kenhy' and 'Johnstone' tall fescue with low (< 1%) infestation rate of Acremonium coenophialum from October 24 to December 19; 2) drylot feeding of Johnstone and HE KY-31 haylage (December 19 to April 10); and 3) feedlot finishing on a common high-concentrate diet (April 11 to August 1). In Phase 1, ADG was greatest (P < .05) for Kenhy, intermediate for Johnstone and LE KY-31, and lowest (P < .05) for HE KY-31. Implantation with estradiol 17-beta increased ADG (P < .01) by 23, 27, 7, and 2% for steers grazing Johnstone, HE KY-31, LE KY-31, and Kenhy, respectively. Dry matter digestibility and DMI of stockpiled Johnstone and HE KY-31 were not different (P > .10). During Phase 2, steers consuming Johnstone haylage had greater (P < .01) DMI, ADG, and gain:feed ratio (G:F) than steers consuming HE KY-31 haylage. During Phase 3, steers previously consuming Johnstone had greater DMI (P < .10); however, steers previously fed HE KY-31 had greater ADG (P < .05) and G:F (P < .01). By the end of the study, steer body weights were not different (P > .10) between treatments. These data indicate that growth-decreasing effects of endophyte-infested fescue were evident at hypothermal-ambient temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)