Determination of cellular uptake and intracellular levels of Cenersen (Aezea(®), EL625), a p53 antisense oligonucleotide in acute myeloid leukemia cells.

摘要: TP53 encodes for tumor protein p53. The suppression of p53 results in interruption DNA repair mechanisms dividing malignant cells thereby increasing the damage and activating p53-independent apoptosis. This ultimately may translate into enhanced cytotoxic effects standard chemotherapy. Based on this rationale, Cenersen, a phosphorothioate oligonucleotide antisense to p53-mRNA was synthesized tested clinical trials patients with acute myeloid leukemia (AML). An important component Cenersen development is develop sensitive specific method quantify plasma intracellular levels different biologic matrices order determine tissue distribution parent compound its metabolites. Ultimately, will allow us pharmacokinetic pharmacodynamic relationship dose-effect correlation design effective regimen be rapidly clinic. ELISA-based assay adapted validation mouse cell lysate. Cellular uptake studied MV4-11 KASUMI-1 AML lines. Real-time RT-PCR used measure P53-mRNA expression changes treated cells. had limit quantification 35pmol/L plasma. Within-day between-day precision <15% accuracy nearly 100% were observed linear range 10-2000pmol/L (R(2)=0.99) selectivity examined as cross-reactivity 3'N-1, 3'N-2-metabolites, 16.8% 0.4%, respectively, mismatch scramble oligonucleotides 0.06% respectively. stable up 8h at 37°C. When exposed 0.1-1μmol/L showed concentration 9.97-45.34nmol/mg 0.1-2.1nmol/mg protein, Successful downregulation applicable measurement Cenersen. Assessment achievable useful elucidate biological activity promising drug define recommended dose future trials.