Abstract 4781: Analysis of a new chemopreventative agent SHetA2 and its metabolites in Beagle dog liver and plasma

Flexible heteroarotinoid SHetA2 {[(4-nitrophenyl)amino][2,2,4,4-tetramethylthiochroman-6-yl)amino] methanethione]} is a new candidate anticancer compound which is effective against a number of different types of tumors. We conducted preclinical safety and toxicity testing of SHetA2 in Beagle dogs with emphasis on determination of SHetA2 and its metabolites (especially GSH-conjugates) in liver. SHetA2 (in 30% aqueous Solutol® HS 15 to improve bioavailability) was administered orally to male and female Beagle dogs for 29 consecutive days. The dose levels of SHetA2 were 0 (control group 1), 100 (group 2), 400 (group 3), and 1500 (group 4) mg/kg/day. The liver and plasma samples were collected on day 29 at 2 hours after drug administration. SHetA2 and its metabolites levels were measured by high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS) on Micromass Quattro micro API triple quadrupole mass-spectrometer. Metabolites formation was also confirmed by high-resolution accurate mass analysis on Waters SYNAPT time-of-flight (TOF) mass spectrometer. The following known metabolites of SHetA2 were quantified in liver (using the parent compound calibration curve): monohydroxy SHetA2 [SHetA2(O1)], dihydroxy SHetA2 [SHetA2(O2)], and GSH-conjugates of SHetA2 [SHetA2-GSH], monohydroxy SHetA2 [SHetA2(O1)-GSH], and dihydroxy SHetA2 [SHetA2(O2)-GSH]. Trihydroxy SHetA2 and its GSH-conjugate were below quantification level or not detected. The GSH-metabolites were of particular interest since conjugation with GSH may serve as a potential indicator of a compound toxicity. However, despite the relatively high levels of SHetA2-GSH-conjugate in dog liver, no drug-related mortality or clinical signs of toxicity were seen in any dose groups. We also found significant levels of several previously unknown metabolites - glucuronide conjugates of SHetA2, SHetA2(O1), and SHetA2(O2). These glucuronides were not reported in mouse and rat liver, in contrast to our dog study. The exact position of glucuronidation was not determined. It may represent a common N- or a rare case of S-glucuronidation for SHetA2, and, in addition, O-glucuronidation for oxidation products of SHetA2. The most abundant metabolites in liver were: glucuronide of SHetA2, SHetA2(O1) and its glucuronide, and GSH-conjugate of SHetA2 whose levels were at 29-80%, 16-31%, 14-39%, and 9-20%, respectively, of SHetA2 levels (2,600-14,000 ng/g; based on average values). Dihydroxy SHetA2 showed two maj