Novel organic nitrate prodrug 4(R)-N-(2-Nitroxyethyl)-2-oxothiazolidine-4-carboxamide (RS-7897) serves as a xenobiotic substrate for pyroglutamyl aminopeptidase I in dogs.

RS-7897, a novel organic nitrate, structurally contains aminoethylnitrate (AEN) and L-2-oxothiazolidine-4-carboxylic acid (L-OTCA), which are linked together via an amide bond. Vasodilating activity of RS-7897 was 130 times weaker than that of AEN in vitro, while in vivo it was comparable to but longer lasting than those of AEN and nitroglycerin in anesthetized dogs. Intravenous administration of RS-7897 to dogs resulted in the appearance in plasma of AEN, which decreased with about 2.5 times longer t(1/2) (0.49 h) than that after administration of AEN itself. The T(max) value of AEN (0.25 h) after RS-7897 dosing agreed with the time showing the maximum vasodilating effect, indicating that RS-7897 serves as a prodrug releasing AEN slowly in vivo. The activity to hydrolyze RS-7897 to AEN and L-OTCA was localized in the cytosolic fractions of dog tissues, inhibited by thiol-blocking agents and was strongly inhibited by thyrotropin-releasing hormone, a substrate of pyroglutamyl aminopeptidase I (PAP-I). Furthermore, the RS-7897-hydrolyzing activity in dog liver cytosol was completely inhibited by an antibody against rat PAP-I. Therefore, it was found that PAP-I is involved in bioactivation of RS-7897 by amide bond hydrolysis, recognizing the sulfur-substituted L-pyroglutamyl moiety (L-OTCA) of this xenobiotic substrate.

Formation of a structurally novel, serial diglucuronide of 4-hydroxybiphenyl by further glucuronidation of a monoglucuronide in dog liver microsomes.

Incubation of 4-hydroxybiphenyl (p-phenylphenol) in the presence of UDP-glucuronic acid (UDPGA) with liver microsomes from male and female dogs produced a more polar metabolite peak than a simultaneously produced peak of 4-hydroxybiphenyl monoglucuronide in the high performance liquid chromatography (HPLC) chromatogram. Tandem mass spectrometry (MS/MS) and two-dimensional nuclear magnetic resonance (NMR) analyses revealed this polar metabolite as a 4-hydroxybiphenyl diglucuronide having a beta-D-glucuronopyranosyl-(1-->2)-beta-D-glucuronopyranosyl moiety, where the two glucuronic acids are connected directly at the 1''-->2' position. Liver microsomes from Sprague-Dawley rat, cynomolgus monkey and human, converted 4-hydroxybiphenyl only to the monoglucuronide, suggesting that there is a dog UDP-glucuronosyltransferase (UGT), with a wider substrate specificity capable of glucuronidating 4-hydroxybiphenyl monoglucuronide to the diglucuronide.