TPN729 is a novel
phosphodiesterase 5 (
PDE5) inhibitor used to treat
erectile dysfunction in men. Our previous study shows that the plasma exposure of metabolite M3 (N-dealkylation of
TPN729) in humans is much higher than that of
TPN729. In this study, we compared its metabolism and pharmacokinetics in different species and explored the contribution of its main metabolite M3 to pharmacological effect. We conducted a combinatory approach of ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry-based metabolite identification, and examined pharmacokinetic profiles in monkeys, dogs, and rats following
TPN729 administration. A remarkable species difference was observed in the relative abundance of major metabolite M3: i.e., the plasma exposure of M3 was 7.6-fold higher than that of
TPN729 in humans, and 3.5-, 1.2-, 1.1-fold in monkeys, dogs, and rats, respectively. We incubated liver S9 and liver microsomes with
TPN729 and
CYP3A inhibitors, and demonstrated that
CYP3A was responsible for
TPN729 metabolism and M3 formation in humans. The inhibitory activity of M3 on PDE5 was 0.78-fold that of
TPN729 (The IC50 values of
TPN729 and M3 for PDE5A were 6.17 ± 0.48 and 7.94 ± 0.07 nM, respectively.). The
plasma protein binding rates of
TPN729 and M3 in humans were 92.7% and 98.7%, respectively. It was astonishing that the catalyzing capability of
CYP3A4 in M3 formation exhibited seven-fold disparity between different species. M3 was an active metabolite, and its pharmacological contribution was equal to that of
TPN729 in humans. These findings provide new insights into the limitation and selection of animal model for predicting the clinical pharmacokinetics of drug candidates metabolized by
CYP3A4.