Study Of Oral Peptidomimetic Prodrugs Of Didanosine

Didanosine (ddI) is nucleoside antiviral drugs and is used to inhibit HIV replication in clinical. However, the great molecular polarity because of sugar group of ddI can cause poor intestinal membrane permeability. And also ddI was very unstable under acidic conditions, for it degrades into no-activie hypoxanthine. So ddI has a low oral bioavailability (about 20～40%). There had been some modifications on the hydroxyl of 5’groups such as anhydride or glyceride to form lipophilic prodrug to improve its oral bioavailability. But as far as we know there was not the report of peptidomimetic prodrugs of ddI. The purpose of paper was to develop the prodrug that could be specifically identified by intestinal PepTl(oligopeptide transporter 1) and increased membrane permeability and chemical stability. So that the prodrug could enhance the oral bioavailability of ddI, raise patient compliance, and reduce individual differences.Six prodrugs of ddI (L-valine, D-valine, L-phenylalanine, D-phenylalanine, L-tryptophan and isobutyrate prodrug) were synthesized through acylation reaction and catalytic hydrogenation. And we optimized the synthetic route for the purpose of increasing the yield of targeted products.The membrane permeability of the six prodrugs and ddI was studied in Caco-2 cells. L-valine ester prodrug (5’-O-L-valyl-ddI) had highest permeability in all prodrugs with 8.74×10-6cm/s, which was the parent drug ddI of 14.3 times. Therefore 5’-O-L-valyl-ddI, was selected as lead compound for further study, such as its uptake mechanism, stability and pharmacokinetics.Gly-Sar (a typical substrate of PepTl) uptake inhibition experiments showed that Gly-Sar uptake could be inhibited by 5’-O-L-valyl-ddI as a concentration-dependent. The half inhibition concentration (50% inhibitory concentration, IC50) was 0.27±0.07 mM. Leptin was used to induce high expression of PepTl in Caco-2 cells. The uptake of 5’-O-L-valyl-ddI in leptin-induced Caco-2 cell was more than in the control Caco-2 cells significantly and the uptake 5’-O-L-valyl-ddI could be significantly inhibited by Gly-Sar, but not by L-valine. In vitro uptake experiments showed that the 5’-O-L-valyl-ddI was a substrate of PepT1. The kinetic analysis of 5’-O-L-valyl-ddI in Caco-2 cells showed that the Km and Vmax of 5’-O-L-valyl-ddI was 0.91 mM and 11.94 nmol/mg protein/10min respectively.The stability experiments were performed in phosphate buffers, rat plasma and tissue homogenates at 37℃. The chemical stability of 5’-O-L-valyl-ddI was notably influenced by the pH value of phosphate buffer. The hydrolysis was catalyzed by H+ or OH-. Compound 5’-O-L-valyl-ddI was more stable at pH 6.0. The t1/2 value of 5’-O-L-valyl-ddI in the hepatic homogenates, plasma and intestinal homogenate was short. This implied that compound 5’-O-L-valyl-ddI could rapidly converted to ddI in vivo. The t1/2 of ddI was very short in simulated gastric juice and could not be detected in two minutes. But 5’-O-L-valyl-ddI could increase ddI stability in simulated gastric juice and its t1/2 was 36 min.When rats were orally administrated with 5’-O-L-valyl-ddI, the AUC and Cmax of ddI gradually increased from 5 to 30 mg/kg. The oral absolute bioavailability of ddI was 47.2% and 7.9% after orally administrated 5’-O-L-valyl-ddI and ddI to rats at a dose of 15 mg/kg. When oral coadministration with PepTl typical substrate, Gly-Sar (100 mg/kg), the absolute bioavailability of ddI following oral administration of compound 5’-O-L-valyl-ddI (10 mg/kg, calculated as ddI dose) decreased from 46.6% to 33.9%, respectively. The results showed the competitive inhibition of Gly-Sar on the PepTl-mediated transport of 5’-O-L-valyl-ddI indeed occurred in vivo, in an agreement with in vitro results. Although the inhibitory effect (27.4%) was not so significant, the result did reflect the inhibitory tendency of Gly-Sar on the oral absorption of compound 5’-O-L-valyl-ddI.The pharmacokinetic experiments in vivo with combination of anti-acid showed that anti-acids could increase ddI oral bioavailability significantly, but the enhancement of 5’-O-L-valyl-ddI, bioavailability was not as distinctly as ddI, which proved that 5’-O-L-valyl-ddI could improve stability of ddI in acid indirectly. The portal vein samples revealed that the majority of compound 5’-O-L-valyl-ddI bioactivation occurred in the intestinal and then the minority in the liver before reaching the systemic circulation, with Cmax value of 5’-O-L-valyl-ddI in portal vein only 10% of ddI in jugular vein, indicating that 5’-O-L-valyl-ddI in the small intestine cells was fully activated and those residue prodrug was activated in the liver rapidly. The bioactivation mechanism study indicated that the first pass effect in the small intestine was likely to be an important way for bioactivation of 5’-O-L-valyl-ddI.