Propylene Glycol-Linked Amino Acid/Dipeptide Diester Prodrugs of Oleanolic Acid for PepT1-Mediated Transport: Synthesis, Intestinal Permeability, and Pharmacokinetics

Abstract

In our previous studies, ethylene glycol-linked amino acid diester prodrugs of oleanolic acid (OA), a Biopharmaceutics Classification System (BCS) class IV drug, designed to target peptide transporter 1 (PepT1) have been synthesized and evaluated. Unlike ethylene glycol, propylene glycol is of very low toxicity in vivo. In this study, propylene glycol was used as a linker to further compare the effect of the type of linker on the stability, permeability, affinity, and bioavailability of the prodrugs of OA. Seven diester prodrugs with amino acid/dipeptide promoieties containing l-Val ester (<b>7a</b>), l-Phe ester (<b>7b</b>), l-Ile ester (<b>7c</b>), d-Val-l-Val ester (<b>9a</b>), l-Val-l-Val ester (<b>9b</b>), l-Ala-l-Val ester (<b>9c</b>), and l-Ala-l-Ile ester (<b>9d</b>) were designed and successfully synthesized. In situ rat single-pass intestinal perfusion (SPIP) model was performed to screen the effective permeability (<i>P</i>_eff) of the prodrugs. <i>P</i>_eff of <b>7a</b>, <b>7b</b>, <b>7c</b>, <b>9a</b>, <b>9b</b>, <b>9c</b>, and <b>9d</b> (6.7-fold, 2.4-fold, 1.24-fold, 1.22-fold, 4.15-fold, 2.2-fold, and 1.4-fold, respectively) in 2-(<i>N</i>-morpholino)­ethanesulfonic acid buffer (MES) with pH 6.0 showed significant increase compared to that of OA (<i>p</i> < 0.01). In hydroxyethyl piperazine ethanesulfonic acid buffer (HEPES) of pH 7.4, except for <b>7c</b>, <b>9a</b>, and <b>9d</b>, <i>P</i>_eff of the other prodrugs containing <b>7a</b> (5.2-fold), <b>7b</b> (2.0-fold), <b>9b</b> (3.1-fold), and <b>9c</b> (1.7-fold) exhibited significantly higher values than that of OA (<i>p</i> < 0.01). In inhibition studies with glycyl-sarcosine (Gly-Sar, a typical substrate of PepT1), <i>P</i>_eff of <b>7a</b> (5.2-fold), <b>7b</b> (2.0-fold), <b>9b</b> (3.1-fold), and <b>9c</b> (2.3-fold) had significantly reduced values (<i>p</i> < 0.01). Compared to the apparent permeability coefficient (<i>P</i>_app) of OA with Caco-2 cell monolayer, significant enhancement of the <i>P</i>_app of <b>7a</b> (5.27-fold), <b>9b</b> (3.31-fold), <b>9a</b> (2.26-fold), <b>7b</b> (2.10-fold), <b>7c</b> (2.03-fold), <b>9c</b> (1.87-fold), and <b>9d</b> (1.39-fold) was also observed (<i>p</i> < 0.01). Inhibition studies with Gly-Sar (1 mM) showed that <i>P</i>_app of <b>7a</b>, <b>9b</b>, and <b>9c</b> significantly reduced by 1.3-fold, 1.6-fold, and 1.4-fold (<i>p</i> < 0.01), respectively. These results may be attributed to PepT1-mediated transport and their differential affinity toward PepT1. According to the permeability and affinity, <b>7a</b> and <b>9b</b> were selected in the pharmacokinetic studies in rats. Compared with group OA, <i>C</i>_max for group <b>7a</b> and <b>9b</b> was enhanced to 3.04-fold (<i>p</i> < 0.01) and 2.62-fold (<i>p</i> < 0.01), respectively. AUC_0→24 was improved to 3.55-fold (<i>p</i> < 0.01) and 3.39-fold (<i>p</i> < 0.01), respectively. Compared to the ethylene glycol-linked amino acid diester prodrugs of OA in our previous work, results from this study revealed that part of the propylene glycol-linked amino acid/dipeptide diester prodrugs showed better stability, permeability, affinity, and bioavailability. In conclusion, propylene glycol-linked amino acid/dipeptide diester prodrugs of OA may be suitable for PepT1-targeted prodrugs of OA to improve the oral bioavailability of OA