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

The purposes of this study were to expand the structure of parent drugs selected for peptide transporter 1 (PepT1)-targeted ester prodrug design and to improve oral bioavailability of oleanolic acid (OA), a Biopharmaceutics Classification System (BCS) class IV drug. Through an ethoxy linker the carboxylic acid group of OA was conjugated with the carboxylic acid group of different amino acid promoieties to form six diester prodrugs. The effective permeability (<i>P</i>_eff) of prodrugs was screened by in situ rat single-pass intestinal perfusion (SPIP) model in two buffers with different pH (6.0 and 7.4) as PepT1 employs a proton-gradient as the driving force. Compared to OA, 2.5-fold, 2.3-fold, 2.2-fold, 2.1-fold, and 1.9-fold enhancement of <i>P</i>_eff in buffer with pH 6.0 was observed for l-Phe ester (<b>5c</b>), l-Val ester (<b>5a</b>), l-Lys ester (<b>5e</b>), d-Phe ester (<b>5d</b>), and d-Val ester (<b>5b</b>), respectively. Furthermore, <i>P</i>_eff of <b>5a</b>, <b>5c</b>, <b>5d</b> and <b>5e</b> in pH 6.0 was significantly higher than that in pH 7.4 (<i>p</i> < 0.01), respectively. These results showed that the H⁺ concentration of perfusion solution had great effect on the transport of the prodrugs across intestinal membrane. For the further evaluation of affinity to PepT1, inhibition studies were performed by coperfusing 0.1 mM prodrug with 50 mM glycyl-sarcosine (Gly-Sar, a typical substrate of PepT1). It turned out that the <i>P</i>_eff of <b>5a</b>, <b>5b</b>, <b>5c</b> and l-Tyr ester (<b>6f</b>) significantly reduced in the presence of Gly-Sar (1.7-fold, 2.2-fold, 1.9-fold, and 1.4-fold, respectively). We supposed that it may be attributed to PepT1 mediated transport of these prodrugs. <b>5a</b> and <b>6f</b> were selected as the optimal target prodrugs for oral absorption <i>in vivo</i>. Following intragastric administration of 300 mg/kg (calculated as OA) <b>5a</b>, <b>6f</b> and OA in three groups of rats, compared with group OA, <i>C</i>_max for the group of <b>5a</b> and <b>6f</b> was enhanced by 1.56-fold and 1.54-fold, respectively. <i>F</i>_app of group <b>5a</b> and <b>6f</b> was 2.21- and 2.04-fold increased, respectively, indicating that <b>5a</b> and <b>6f</b> had better oral absorption than OA. The combined results also suggest that diester prodrugs which conjugated two carboxylic acid groups of proper amino acid promoieties and parent drug through a linker can be used for PepT1-targeted prodrug design. With this strategy, oral bioavailability of OA in rats could be improved significantly

Discovery of a Potential Anti-Inflammatory Agent: 3‑Oxo-29-noroleana-1,9(11),12-trien-2,20-dicarbonitrile

Fifteen novel derivatives of glycyrrhetinic acid (GA) were synthesized and evaluated for anti-inflammatory activities. It was found that the introduction of 1-en-3-one and 9(11),12-diene and 2,20-dinitrile functionalities into the scaffold of GA led to the discovery of potent compound <b>19</b> for inhibition of LPS-induced NO production. Furthermore, <b>19</b> effectively inhibited the protein and mRNA expression of inducible NO synthase (iNOS) and the mRNA expression of TNF-α, IL-6, and IL-1β in LPS-stimulated RAW 264.7 macrophages. Mechanistically, <b>19</b> exerted inhibitory effects on the activation of the three main MAPKs and phosphorylation and degradation of IκB-α, as well as the ratio of nuclear/cytosolic content of p65. Importantly, <b>19</b> significantly decreased the mortality rate in the mouse model of LPS-induced sepsis shock. It is noteworthy that inhibitory effect of <b>19</b> on NO production was not blocked by the glucocorticoid receptor antagonist mifepristone, indicating that it does not act through the glucocorticoid receptor