| Antisense oligonucleotides (ASONs) are new biological technical agents, designed to block the gene function by selective inhibit the expression of specific genes. ASONs are being explored as potential therapeutics against viral infections, cardiovascular disease, and cancer. Cantide and Flutide are 20-mer and 13-mer antisense phosphorothioate oligonucleotides, which showed significant antitumor activities and antivirus activites in vitro and in vivo. The works of this paper was designed to optimize the analytical methods of qualitiy control, and as well to evaluate the pharmacokinetic features of Cantide and Flutide in animals, so as to support the pharmacokinetic study in human. Some conclusions were taken in the below:1. The establishments of analytical methods about antisense phosphorothioate oligonucleotidesCapillary gel electrophoresis (CGE) is an effective tool for the analysis of antisense oligonucleotides. Separation of full length N and N-1 deletion analogs were achieved (R>1.5) by operating at the gel concentration, column temperature and voltage. Fully thiolated oligonucleotides and partial phosphodiesters analogs (P=O)x can be separation completely by optimized anion-exchange HPLC. The lowest detectable limit of quantification of single phosphorothioater analogs (P=O)? was 1%. 31P-NMR was used to analysis the contents of trace amount phosphodiesters analogs. In this study, we developed a protocol that applied Sanger sequencing reactions in combination with MALDI TOF-MS to sequence Cantide and Flutide.2. The preclinical Pharmacokinetics study of CantideA sensitive and specific CGE method with UV detection for the quantification of Cantide in mouse plasma, urine, feces and rat bile was developed and evaluated. Four standard curves were generated with the internal standard concentration hold at 25.0μg/ml. The linear regions are 2-800μg/ml, 1-160μg/ml, 1-160μg/g and 2-200μg/ml in mouse plasma, urine, feces and rat bile respectively. In plasma, relative standard deviations (RSDs) % of intra-day and inter-day were 2.35%-15.94% and 1.30%-6.85%; the mean relative recovery was between 97.42%±4.20% and 104.45%±16.64%. In urine, RSDs of intra-day and inter-day were 1.26%-15.13% and 0.90%-2.94%; the relative recovery was between 96.53%±1.66% and 106.54%±12.22%. In feces, RSDs of intra-day and inter-day were 1.53%-12.97% and 0.43%-3.75%; the relative recovery was between 90.92%±1.39% and 110.75%±7.79%. In rat bile, RSDs of intra-day and inter-day were 3.76%-9.04% and 1.01%-2.54%; the relative recovery was between 96.64%±6.51% and 99.20%±6.59%%. CGE analysis of intact drug levels in biological samples provided sufficient sensitivity to support pharmacokinetic evaluations for Cantide.Following single i.v. administration with three levels of dosage of Cantide (25, 50 and 100mg/kg), the plasma samples were collected at different time after dosing and determined by CGE method described as above. Pharmacokinetic parameter modeling and pharmacokinetic calculations were carried out using DAS software and statistical moments respectively. It was shown that the concentration-time data was in accord with two-compartment open pharmacokinetic model in mice after i.v. administration. The mean residence times of three levels of dosage of Cantide were 7.99, 17.07 and 17.52min respectively. Their half lives were 9.68, 27.01 and 33.55min, and AUCs were 1017.28, 2570.68 and 4366.79mg/L·min. The results showed that Cantide was rapidly distributed and eliminated in mice.The excretion properties of Cantide were determined after i.v. (intravenous) administration of a single dose to mice and rat. 7.65% of the delivered dose was excreted over 48h after i.v. dose. Most (6.26%) of Cantide and its metabolites was excreted in the urine and very little (1.39%) appeared in the feces. There was absence in the bile. The greatest percentage of the excreted oligonucleotides in urine and feces was generally in the form of full-length sequence, the kinds of the excreted oligonucleotides was basically same at different duration over 48h.In vitro binding of Cantide to human, dog, rat plasma protein and HAS was determined by ultrafiltration methods. Cantide was shown to be highly bound (>99%) across species (human, dog, rat) and HAS (>93%). The results indicated that Cantide was primarily bound to HAS in human plasma, and it appeared the saturation with the increasing of drug concentration.3. The Pharmacokinetics and bioavaiabilidy studies of Flutide CGE was applied to research the characteristics of Pharmacokinetics in mice after single i.v. and i.n. (intranasal) administrations with 40mg/kg Flutide. Pharmacokinetic parameter modeling and pharmacokinetic calculations were carried out using DAS software and statistical moments. It was shown that the concentration-time data was fit to two-compartment and one-compartment open pharmacokinetic models respectively after i.v. and i.n. administrations. The mean residence times of Flutide after i.v. and i.n. administrations were 17.33min and 94.04min. Their peak concentrations were 425.89 and 11.16mg/L respectively. The results indicated that Flutide will be rapidly distributed and eliminated in mice after i.v. administration, but its duration was long after i.n. administration.Following i.n. administration to mice, the absolution bioavaiability of Flutide is 51.48%. And the distribution of Flutide in lung tissue following i.v. was far higher than that following i.v. administration. The highest concentration in lung, 1099.36±393.00μg/g, was found at 30min after i.n. administration, whereas the peak concentration after i.v. administration was 8.61±4.27μg/g. In summary, the studies demonstrated that intranasal administration was an effective local drug delivery mode.
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