This dissertation is the investigation of a prodrug strategy for increased oral absorption via transport by hPEPT1 and activation by hVACVase. L-Valine, L-isoleucine and L-phenylalanine esters of a guanidino-containing model compound [3-(hydroxymethyl)phenyl]guanidine (3-HPG) were synthesized and evaluated for the feasibility of this prodrug approach to guanidino-containing drugs. In HeLa/hPEPT1 cells, Val-3-HPG and Ile-3-HPG exhibited high affinity to hPEPT1 (IC50: 0.65 mM and 0.63 mM, respectively), and all three L-amino acid esters showed higher uptake (2.6- to 9-fold) than the parent compound 3-HPG. Val-3-HPG and Ile-3-HPG demonstrated remarkable Caco-2 permeability enhancement, and Val-3-HPG exhibited comparable permeability to valacyclovir. The low permeability of D-Val-3-HPG and the inhibition studies indicated that hPEPT1 was the predominant transporter responsible for Val-3-HPG permeability. In rat perfusion studies, Val-3-HPG and Ile-3-HPG permeabilities were significantly higher than 3-HPG, and exceeded/matched the high-permeability standard metoprolol, respectively. In HeLa and Caco-2 cell homogenates, the hydrolysis of amino acid esters was faster than in the corresponding buffer, indicating predominant enzymatic activation. All the L-amino acid 3-HPG esters were found to be good substrates of hVACVase, and exhibited higher specificity constants than that for valacyclovir (kcat/Km in mM-1•s-1: Val-3-HPG, 3370; Ile-3-HPG, 1580; Phe-3-HPG, 1660; valacyclovir, 850), presumably resulting from high binding affinity to hVACVase as well as favorable leaving group lability. The results in this study suggested that the leaving group had an effect on both binding and specific activity of hVACVase-catalyzed prodrug activation. hVACVase is an ideal target for α-amino acid ester prodrugs with relatively labile leaving groups but is unable to activate amide prodrugs
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