Design And Evaluation Of Oral Peptidomimetic Prodrugs Of Decitabine

Decitabine (DAC) is nucleoside anti-neoplastic drugs and it was approved by the FDA for the treatment of myelodysplastic syndrome (MDS) in May2006. To date, it has been the most extensively and advanced investigated DNA methyltransferase (DNMT) inhibitor although there are abundant DNMT inhibitors undergoing preclinical and clinical evaluation. In vivo, DAC is phosphorylated by deoxycytidine kinase to form an active metabolite,5-azadeoxycytidine triphosphate, which is then incorporated into DNA strands and prevents DNA methylation. However, the great molecular polarity of DAC can cause poor intestinal membrane permeability. Due to rapid deamination to the biologically inactive5-aza-2’-deoxyuridine in intestinal and hepatic cells, DAC exhibits a very short plasma half-life and a very low oral bioavailability (about9%), which necessitates the continuous infusion to maintain therapeutical plasma level clinically. But as far as we know there was no report of peptidomimetic prodrugs of DAC. The purpose of paper was to develop the prodrug that could be specifically identified by intestinal PepTl (oligopeptide transporter1) and increase membrane permeability. So that the prodrug could enhance the oral bioavailability of DAC, raise patient compliance and reduce individual differences.Five amino acid prodrugs of DAC (L-valine, D-valine, L-isoleucine, L-phenylalanine and L-tryptophan prodrug) were synthesized through acylation reaction and catalytic hydrogenation. And we optimized the synthetic route and purification method for the simple and practicable reaction condition and increasing the yield of targeted products.The transport characteristics of the five prodrugs were studied in Caco-2cells to screen the target compound with high permeability. Among the compounds,5’-L-valyl-dac (9.94X10-7cm/s) and5’-O-L-phenylanlanyl-dac(1.13×10-6cm/s) possess higher permeability, which were3.28and3.75times as high as DAC, respectively. Meanwhile, based on high permeability and different amino acid promoieties,5’-O-L-valyl-dac (aliphatic) and5’-O-L-phenylanlanyl-dac (aromatic) were selected as the lead compounds for further study, such as uptake mechanism, stability and in vivo pharmacokinetics.Gly-Sar (a typical substrate of PepT1) uptake inhibition experiments indicated that Gly-Sar uptake could be inhibited by5’-O-L-valyl-dac and5’-O-L-phenylanlanyl-dac as a concentration-dependent. The half inhibition concentration (50%inhibitory concentration, IC50) were2.20±0.28mM and3.46±0.16mM, respectively. DAC did not exhibit any inhibition at all the tested concentrations. Leptin was used to induce high expression of PepTl in Caco-2cells. And the uptake of Gly-Sar by the leptin-treated Caco-2cells was compared with the control Caco-2cells to evaluate whether this treatment was successful. The uptake of5’-O-L-valyl-dac and5’-O-L-phenylanlanyl-dac in leptin-induced Caco-2cell was more than in the control Caco-2cells significantly, while the uptake of DAC are not significantly different between the leptin-treated and control Caco-2cells. In vitro uptake experiments showed that the two compounds were substrates of PepTl, while DAC lacked any apparent affinity for the transporter.The stability experiments were performed in phosphate buffers of different pH values, rat tissue homogenates, plasma, gastric and intestinal fluids at37℃. The chemical stability of5’-O-L-valyl-dac and5’-O-L-phenylanlanyl-dac was notably influenced by the pH value of phosphate buffer. The chemical stability of two prodrugs basically decreased with the increasing pH of phosphate buffer. The t1/2values of5’-O-L-valyl-dac in the intestinal homogenates, hepatic homogenates and plasma were61,5and86min, respectively. The ti/2values of5’-O-L-phenylanlanyl-dac in the intestinal homogenates and hepatic homogenates were7and2min, the half-life of the compound in plasma was extremely short and5’-O-L-phenylanlanyl-dac could not be detected. In the rat gastric fluids and intestinal fluids, the half-lives of the two compounds both were greater than1.5h. This implied that the two compounds could maintain enough chemical stability in gastrointestinal tract and was rapidly converted to active parent drug by ester enzymes following PepTl-mediated transport across the intestinal membrane.5’-O-L-phenylanlanyl-dac had a higher in vivo bioconversion rate compared to5’-O-L-valyl-dac.The oral absolute bioavailability of DAC following oral administration of5’-O-L-valyl-dac,5’-O-L-phenylanlanyl-dac and DAC to rats at a dose of15mg/kg (calculated as DAC dose) was46.7%,50.9%and26.9%, respectively. When oral coadministration with PepTl typical substrate, Gly-Sar (150mg/kg), the absolute bioavailability of DAC following oral administration of prodrug to rats decreased from46.7%to32.2%for5’-O-L-valyl-dac and from50.9%to32.5%for 5’-O-L-phenylanlanyl-dac, respectively.