Preclinical Research Of Phosphorothioate Antisense Oligonucleotide Named "Cantide"

Cantide is a 20-mer antisense phosphorothioate oligonucleotides targeted to human telomerase reverse transcriptase (hTERT) mRNA. Cantide has significant antitumor activities in vitro and in vivo, providing a new antisense drug candidate for cancer gene therapy. Here the researches are mainly about synthesis and purification, evaluation of in vitro antitumor activities and pharmacokinetics of Cantide. All these researches will promote the novel drug candidate to clinical trail.Firstly, the synthesis and purification scales were enlarged successfully. Cantide was synthesized with T-supporter using the standard "DMT-ON" protocols of AKTA oligopilot II .The method was finally scaled up to 2mmol. Purification utilized the hydrophobicity of the dimethoxytrityl (DMT) protecting group and the hydrophobicity of SOURCE 15Q media. The oligonucleotide with DMT could be highly retained by the SOURCE 15Q. "Trityl off' failure sequences were easily separated from "trityl on" oligmer when high-salt buffer was used firstly. And then the trityl group was cleaved from the product with 0.4% trifluoracetic acid. In the end, a linear gradient was used to separate full-length, full thioated phosphorothioate contents from synthetic failure products. UV absorption wavelength was selected at 290nm, linear flow rate was 382cm/h and sample load was 12mg/g media. We developed a single step anion-exchange chromatographic protocol for purifying Cantide at a final purity of 98.23%.Secondly, the inhibitory effect of Cantide on twelve human cells proliferation was estimated by MTS assay in vitro. In a 96 culture plate, 3,000⁴,000 cells were seeded in 200μl of the appropriate growth medium supplanted with serum. After incubating the cells for 24h at 37℃ in a CO₂ incubator, Lipofectin Reagent was used to transfect Cantide with different concentrations. Following the transfection, the DNA-containing...