Transporter-Targeted Prodrug Delivery to Improve Oral Bioavailability of Saquinavir

Abstract

Saquinavir (SQV) is the first antiretroviral protease inhibitor approved by Food and Drug Administration in the United States for the treatment of HIV infection due to its potent anti-HIV activity. However, some unfavorable properties including low aqueous solubility, low intestinal absorption and fast biotransformation lead to its poor oral bioavailability and limited therapeutic efficacy. The objective of this dissertation project is to investigate whether stereoisomerized peptide prodrugs targeting influx transporter system could improve intestinal absorption and oral bioavailability of SQV. Four stereoisomeric dipeptide prodrugs of SQV including L-valine-L-valine-SQV (LLS), L-valine-D-valine-SQV (LDS), D-valine-L-valine-SQV (DLS) and D-valine-Dvaline- SQV (DDS) and two amino acid prodrugs including L-valine-SQV (LS) and Dvaline- SQV (DS) were synthesized and investigated using in vitro cell culture models. All dipeptide prodrugs exhibit improved aqueous solubility, lowered cytotoxicity, and reduced P-gp/MRP2-mediated efflux activities regardless of stereochemistry in promoieties. SQV attached with L-isomers shows higher affinity for peptide transporters but lower stability and higher toxicity, whereas conjugation with D-isomers can enhance stability and reduce toxicity, but not be recognized by peptide transporters. Subsequently, results of metabolism studies performed in rat liver microsomes indicate that elimination half life of SQV was prolonged dramatically by 7- to 40-fold due to prodrug modification with the rank order of DDS > DLS > LDS > LLS > DS > LS > SQV. In comparison with D-configuration, L-configuration favors the interaction between prodrugs and rat hepatic CYP3A enzymes, resulting in a relatively rapid clearance by CYP3A. Stereoselectivity is also observed in protein binding of prodrugs in rat plasma. Lower protein binding was obtained by LS, LLS and LDS but higher by DS, DDS and DLS as compared with SQV. Pharmacokinetics studies performed in rats provide further evidences that oral bioavailability of SQV is drastically enhanced by conjugation with dipeptide L-valine-Dvaline. In conclusion, stereoisomeric dipeptide prodrug modification targeting specific influx transporters could be a successful strategy to improve bioavailability of poorly absorbed drug SQV. Additionally, influence of exogenous human efflux transporters (P-gp, MRP2 and BCRP) on functional activities of endogenous peptide transporters (PepT) was also delineated in MDR-transfected MDCK cell lines in this project. Results demonstrate that overexpression of MDR genes reduces PepT function probably due to the phenomenon of transporter-compensation resulting in down-regulation of endogenous genes. It may provide some mechanistic insight into possible reasons for underestimation in drug screening using these cell models.