Bifunctional Peptidomimetic Prodrugs of Didanosine for Improved Intestinal Permeability and Enhanced Acidic Stability: Synthesis, Transepithelial Transport, Chemical Stability and Pharmacokinetics

Abstract

Five peptidomimetic prodrugs of didanosine (DDI) were synthesized and designed to improve bioavailability of DDI following oral administration via targeting intestinal oligopeptide transporter (PepT1) and enhancing chemical stability. The permeability of prodrugs was screened in Caco-2 cells grown on permeable supports. 5'-O-L-valyl ester prodrug of DDI (compound 4a) demonstrated the highest membrane permeability and was selected as the optimal target prodrug for further studies. The uptake of glycylsarcosine (Gly-Sar, a typical substrate of PepT1) by Caco-2 cells could be inhibited by compound 4a in a concentration-dependent manner. The Caco-2 cells were treated with 0.2 nM leptin for enhanced PepT1 expression. The uptake of compound 4a was markedly increased in the leptin-treated Caco-2 cells compared with the control Caco-2 cells, both of which were obviously inhibited by 20 mM Gly-Sar. The K(m) and V(max) values of kinetic study of compound 4a transported by PepT1 in Caco-2 cells were 0.91 mM and 11.94 nmol/mg of protein/10 min, respectively. The chemical stability studies were performed in simulated gastric fluid (SGF), phosphate buffers under various pH conditions, rat tissue homogenates and plasma at 37 °C. The concentrations of DDI could not be detected in the two minutes in SGF. But compound 4a could significantly increase DDI acidic stability, and its t(½) was extended to as long as 36 min in SGF. Compound 4a was stable in pH 6.0 phosphate buffer but could be quickly transformed into DDI in plasma and tissue homogenates. The oral absolute bioavailability of DDI was 47.2% and 7.9% after compound 4a and DDI were orally administered to rats at a dose of 15 mg/kg, respectively. The coadministration with antiacid agent could also suggest that compound 4a was more stable under harsh acidic conditions compared with DDI. Compound 4a bioavailability in rats was reduced to 33.9% when orally co-administered with Gly-Sar (100 mg/kg). The In Vivo bioactivation mechanism of compound 4a was investigated by comparing the levels of DDI and compound 4a in the jugular and portal veins in rats. The plasma concentration of intact compound 4a was very low in portal veins and could hardly be detected in the jugular vein. In conclusion, compound 4a could significantly improve the oral bioavailability of DDI in rats through PepT1-mediated absorption and enhanced acidic stability, followed by rapid and mostly intracellular bioactivation, the majority in the intestinal cells but the minority in the liver. Additionally, the prodrug strategy targeted to intestinal PepT1 could offer a promising strategy to improve oral bioavailability of poorly absorbed didanosine.

Significance of Peptide Transporter 1 in the Intestinal Permeability of Valacyclovir in Wild-Type and PepT1 Knockout Mice

“…Various PepT1 targeted prodrugs, which are commonly synthesized by linking poorly permeable active drugs with some amino acid or dipeptide carriers, are under intensive investigation (Tsume et al, 2008;Sun et al, 2010;Gupta et al, 2011;Yan et al, 2011). Among these prodrugs, valacyclovir is viewed as the prototype compound with a large available database of mechanistic, absorption, and disposition properties.…”

A Minireview: Usefulness of Transporter-Targeted Prodrugs in Enhancing Membrane Permeability

“…Peptidemimetic prodrugs of DDI were synthesized to increase the oral bioavailability by targeting intestinal PEPT1. 67 Val-DDI exhibited higher permeability than DDI in Caco-2 cells and inhibited the uptake of Gly-Sar in a concentration-dependent manner. DDI was almost completely degraded in simulated gastric fluid within 2 min, but Val-DDI increased the stability of DDI in simulated gastric fluid (half life was 36 min).…”

Spray-dried didanosine-loaded polymeric particles for enhanced oral bioavailability

“…Immediately after extraction, each aliquot (1 mL) was neutralized with 1 N NaOH (150 L) to prevent further degradation of ddI. Then, ddI was quantified by High Performance Liquid Chromatography (Alliance HPLC, separation module e2695, Waters Corp.) [46] using a Waters 5 m, C18, 150 mm × 4.6 mm column (Waters) with a dual-wavelength UV detector ( = 249 and 254 nm, 2998 Photoiodide Array UV/Vis 2D detector, W2998, Waters). For this, a method previously validated for ddI was used with slight modifications [14].…”

“…Both ddI and hypoxanthine were detected at different retention times. The apparent first-order degradation rate constant of ddI was determined by plotting the logarithm of remaining ddI as a function of time [46]. All assays were performed in triplicate and results are expressed as the mean ± S.D.…”

“…Since the solubility of ddI in water is high (∼30 mg/mL) in a broad pH range, a strong influence of the pH on the release rate was not anticipated. In addition, the degradation in acid medium is extremely fast (in the order of few minutes) [46]. In this framework, assays were conducted under conditions that ensured the chemical integrity of ddI during the assay.…”