In Vitro Metabolism of N-(5-Chloro-2-methylphenyl)-N‘-(2-methylpropyl)thiourea:  Species Comparison and Identification of a Novel Thiocarbamide−Glutathione Adduct

Abstract

The in vitro metabolism of SDZ HDL 376, a thiocarbamide developed for the treatment of atherosclerosis, was investigated in rat, dog, monkey, and human liver microsomes, as well as in rat and human liver slices. [14C]SDZ HDL 376 was extensively metabolized in all the species except human. In rat liver microsomes an S-oxide was the major metabolite. In human and monkey microsomes, carbon hydroxylation was favored. The NADPH-dependent oxidation of SDZ HDL 376 resulted in covalent binding to microsomal protein. Addition of GSH to the incubations decreased protein binding in a concentration-dependent manner and resulted in a novel SDZ HDL 376-GSH adduct. Adduct formation required NADPH and was mediated predominantly by cytochrome P450. Inhibition of cytochrome P450 by 1-aminobenzotriazole resulted in a 95% decrease in adduct formation, while heat inactivation of flavin-containing monooxygenases resulted in a 10% decrease. Unlike other thiocarbamides which form disulfide adducts with GSH, the SDZ HDL 376 adduct contained a thioether linkage as characterized by LC/MS/MS and reference to a synthetic standard. Reactions performed with [35S]GSH resulted in a [35S]SDZ HDL 376-GSH adduct, demonstrating the sulfur was derived from GSH. Adduct formation was faster in rat microsomal reactions compared to human microsomes. Other structurally unrelated thiocarbamides (phenylthiourea, methimazole, 2-mercaptobenzimidazole, 2-mercaptoquinazoline, and 2-propyl-6-thiouracil) did not form similar adducts in rat liver microsomes supplemented with GSH. Therefore, the GSH adduct of SDZ HDL 376 is unique for this type of thiocarbamide. These results suggest that the bioactivation and detoxification of SDZ HDL 376 differ significantly from other thiocarbamides. Furthermore, the in vitro formation of S-oxides and GSH adducts in rat hepatic tissue, and ring hydroxylation and glucuronidation in human hepatic tissue, suggests rats may be more susceptible to the toxicity of SDZ HDL 376 compared to humans.

LC-MS/MS identification of the principal in vitro and in vivo phase I metabolites of the novel thiosemicarbazone anti-cancer drug, Bp4eT

“…The oxidative desulfuration of the thiourea structural moiety (R-NH-C(0S)-NR 2 ) leading to the formation of a formamidine structure (i.e., R-N0CH-NR 2 ) is a well-known process already described by Walter et al [30]. It occurs both upon hydrogen peroxide treatment [30] or enzymatically [31].…”

Ethionamide activation and sensitivity in multidrug-resistant Mycobacterium tuberculosis

“…It has long been known that in vivo ETA demonstrates more consistent antitubercular activity than one would expect from in vitro examination of its minimal inhibitory concentration (MIC), and it has been demonstrated that a major metabolite of ETA in vivo is its S-oxide (2), which shows comparable in vitro activity (18)(19)(20)(21)(22). A wide variety of toxic thiocarbonyl-containing compounds have been shown to be converted to the corresponding S-oxides during in vivo metabolism by mammals supporting this as an intermediate oxidation in the metabolic activation of ETA (23).…”

Metabolism of the Antituberculosis Drug Ethionamide

“…A wide variety of toxic thiocarbonyl-containing compounds have been shown to be converted to the corresponding S-oxides during in vivo metabolism by mammals supporting this intermediate oxidation in the metabolic activation of ETH [43]. .…”