The potent, functional agonist of the bile acid Takeda G-protein-coupled receptor 5 (TGR5), (S)-1-(6-fluoro-2-methyl-3,4-dihydroquinolin-1(2H)-yl)-2-(isoquinolin-5-yloxy)ethanone (3), represents a useful tool to probe in vivo TGR5 pharmacology. Rapid degradation of 3 in both rat and mouse plasma, however, hindered the conduct of in vivo pharmacokinetic/pharmacodynamic investigations (including plasma-free fraction (f(u plasma)) determination) in rodent models of pharmacology. Studies were therefore initiated to understand the biochemical basis for plasma instability so that appropriate methodology could be implemented in in vivo pharmacology studies to prevent the breakdown of 3. Compound 3 underwent amide bond cleavage in both rat and mouse plasma with half-lives (T(1/2)) of 39 + or - 7 and 9.9 + or - 0.1 min. bis(p-nitrophenyl) phosphate (BNPP), a specific inhibitor of carboxylesterases, was found to inhibit hydrolytic cleavage in a time- and concentration-dependent manner, which suggested the involvement of carboxylesterases in the metabolism of 3. In contrast with the findings in rodents, 3 was resistant to hydrolytic cleavage in both dog and human plasma. The instability of 3 was also observed in rat and mouse liver microsomes. beta-Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-dependent metabolism of 3 occurred more rapidly (T(1/2) approximately 2.22-6.4 min) compared with the metabolic component observed in the absence of the co-factor (T(1/2) approximately 89-130 min). Oxidative metabolism dominated the NADPH-dependent decline of 3, whereas NADPH-independent metabolism of 3 proceeded via simple amide bond hydrolysis. Compound 3 was highly bound (approximately 95%) to both dog and human plasmas. Rat and mouse plasma, pre-treated with BNPP to inhibit carboxylesterases activity, were used to determine the f(u plasma) of 3. A BNPP concentration of 500 microM was determined to be optimal for these studies. Higher BNPP concentrations (1000 microM) appeared to displace 3 from its plasma protein-binding sites in preclinical species and human. Under the conditions of carboxylesterases-inhibited rat and mouse plasma, the level of protein binding displayed by 3 was similar to those observed in dog and human. In conclusion, a novel system has been devised to measure f(u plasma) for a plasma-labile compound. The BNPP methodology can be potentially applied to stabilize hydrolytic cleavage of structurally diverse carboxylesterase substrates in the plasma (and other tissue), thereby allowing the characterization of pharmacology studies on plasma-labile compounds if and when they emerge as hits in exploratory drug-discovery programmes.