Species-specific in vivo exposure assessment and in vivo-in vitro correlation of the carboxylate esters prodrug HD56 targeting FK506 binding proteins: The pivotal role of humanized mice.

HD561, which was designed to enhance nerve growth, was re-engineered into HD56, a carboxylic acid ester prodrug. The goal of this study was to compare the druggability, species differences, and the correlation between in vitro and in vivo transformation of HD56 to HD561 from a pharmacokinetic (PK) perspective, offering a scientific basis for HD56's clinical research. The bidirectional transmembrane transport of HD56 and HD561 was investigated using Caco-2 cells and LLC-PK1 cells overexpressing MDR1 monolayer cells. Recombinant enzymes and chemical inhibition methods were employed to identify the reaction phenotyping. The conversion of HD56 to HD561 was compared in hepatic and intestinal microsomes, as well as plasma, across different species, including humans, rats, monkeys, and mice with humanized liver. PK studies were conducted in rats, monkeys, and mice with different humanized liver proportions (Hu-URG, Hu-URG-Low, and Hu-URG-High). Finally, an in vivo-in vitro correlation was established between the conversion rate of HD56 to HD561. Results showed that HD56 had better permeability than HD561. HD56 could be hydrolyzed by carboxylesterase 1 to HD561 and be metabolized by cytochrome P450 isoenzymes, while HD561 underwent further metabolism via CYP2C9. Significant species differences existed, and a good in vivo-in vitro correlation was only achieved in humanized mice (r = 0.98). Both in vitro and in vivo PK characteristics of HD56 were remarkably superior to those of HD561, suggesting that HD56 held promise for development. Humanized liver mice serve as a powerful model to address the issue of species differences in ester prodrugs. SIGNIFICANCE STATEMENT: Prodrug HD56 showed superior pharmacokinetic properties compared with the active compound HD561, guiding similar prodrug research. The use of chimeric mice with human hepatocytes, for the first time, to study carboxylesterase (CES) prodrug HD56 provides a model that closely mimics human metabolism. Findings deepen understanding of HD56's behavior and offer a predictive tool for CES prodrugs' metabolic fate, streamlining drug development and improving preclinical accuracy.