• Hai-Feng Song, Zhong-Ming Tang, Shou-Jun Yuan, Bao-Zhen Zhu, and Xiu-Wen Liu.
• Department of Pharmacology and Toxicology, Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China. songhf@nic.bmi.ac.cn
• Acta Pharmacol. Sin. 2003 Mar 1; 24 (3): 269-76.

AimTo optimize the antisense drug design by the combined method of phylogenetic analysis and secondary structure prediction and to get ideal candidates.MethodsThe phylogenetic analysis and the secondary structure simulation were performed by computer. Oligodeoxynucleotides (ODN) were designed against the full-conserved blocks with low local reaction free energy of protein kinase C (PKC)-alpha mRNA. The in vitro effects of ODN were evaluated by human A549 lung carcinoma cells and mouse B16-BL6 melanoma cells, the expression of target mRNA was detected by in situ hybridization and RT-PCR. The in vivo effects of ODN were also evaluated by models of A549 xenografts in nude mice and B16 melanoma in mice.ResultsThree ODN had significantly lower IC50 values than that of ISIS3521, the positive control, on A549 cells in vitro. Five ODN inhibited the growth of B16-BL6 cells with IC50 <100 nmol/L, while IC50 of ISIS3521 was >200 nmol/L. In situ hybridization and RT-PCR showed that the best candidate AP1261 inhibited the expression of PKC-alpha at mRNA level in a dose-dependent manner. AP1261 inhibited the growth of A549 and B16 tumors in vivo at 0.005-0.5 mg.kg(-1).d(-1). The inhibitory rate of AP1261 on A549 tumors was greater than that of ISIS3521 at the same dose. ISIS3521 did not affect the growth of B16 tumors.ConclusionAP1261 may be of value as an antitumor agent or adjuvant and the combined method of phylogenetic analysis and secondary structure prediction is a potential helpful tool for antisense drug design.

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