Plasma Protein Binding, Biostability, Metabolite Profiling, and CYP450 Phenotype of TPB15 Across Different Species: A Novel Smoothened Inhibitor for TNBC Therapy

<b>Background/Objectives</b>: Triple-negative breast cancer (TNBC) is a major cause of cancer-related deaths among women. The Hedgehog (Hh) signaling pathway plays a critical role in tumor development, and targeting this pathway may provide new therapeutic opportunities for TNBC. TPB15 is a novel smoothened inhibitor of the Hh pathway, showing promising tumor reduction and low-toxicity properties in vivo/vitro. This study aims to evaluate TPB15's protein binding rates, metabolic stability, and metabolism across different species, including mice, rats, dogs, monkeys, and humans. <b>Methods</b>: TPB15 was synthesized, and its pharmacokinetic profile was assessed. Plasma protein binding was determined using ultrafiltration across multiple species. Stability studies were conducted in plasma and liver microsomes from each species. Additionally, metabolic enzymes in human liver microsomes were characterized with selective CYP450 inhibitors, and high-resolution mass spectrometry was employed to identify metabolites. <b>Results</b>: Plasma protein binding of TPB15 was consistent across species, ranging from 81.5% to 82.4% in humans and rats. After 120 min, TPB15 remained stable in plasma, with retention rates of 97.2-98.3%. The elimination half-life (<i>t</i>_1/2) varied from 88 min in monkeys to 630 min in dogs. In human liver microsomes, metabolism was significantly inhibited by sulfaphenazole and ketoconazole, indicating the involvement of CYP3A4 and CYP2C9 enzymes. TPB15 underwent phase I metabolism, producing a major metabolite with a molecular weight of 468.9. <b>Conclusions</b>: TPB15 demonstrates stable pharmacokinetic properties across species, with consistent protein binding and significant variability in half-life. The observed differences in metabolism are primarily attributed to CYP2C9 and CYP3A4, offering valuable insights into its drug development potential.