A validated ultrasensitive hybridization-ELISA assay for synthetic microRNAs in mouse plasma and human leukemia cells and its mouse pharmacokinetics
Authors: Zhiliang Xie, Zhongfa Liu, Ming Chiu, Sarah Dunkerson, Natarajan Muthusamy, Ramiro Garzon, Shujun Liu, Carlo Croce, John Byrd, Guido Marcucci, Kenneth Chan
Journal: Cancer Research (2008)

4418  MicroRNAs (miRNAs) are endogenous small non-coding RNAs that bind to target mRNAs and regulate their expression. Recent evidence has suggested the miRNAs involvement in chronic lymphocytic leukemia (CLL) transformation and non-small-cell lung cancer (NSCLC). Aberrantly downregulated miRNAs in malignant cell may be replaced with synthetic miRNAs which may therefore represent novel anticancer therapeutics. Purpose: To develop a sensitive and specific assay to quantify synthetic O-methylated (O-Me) phosphorothioate (PS) miRNAs in biological matrices, to determine their plasma and intracellular concentrations and to conduct their preclinical pharmacological studies. Method: The assay is based on a two-step hybridization technique, with synthetic miRNAs binding to a biotin labeled 9-mer longer capture probe followed by ligation with a 9-mer digoxigenin (Dig) detection probe complementary to the 9-mer overhang of the capture probe. The Dig was detected by anti-Dig-alkaline phosphatase system using fluorescence. The assay was validated in mouse plasma and human leukemia cancer cell lysate using a synthetic O-Me PS miR-29b as a proof of principle. Pharmacokinetics (PK) of O-Me PS miR-29b was conducted in C57BI/6 mice, following its i.v bolus dose of 7.5 mg/kg. The blood samples were collected and peripheral blood cells (PBC) and plasma were separated according to the time schedule of 0.08, 0.17, 0.25, 0.5, 1, 2, 4, 7, 24 h after dosing. The bone marrow (BM) was collected at 2, 4, 7 and 24 h. O-Me PS miR-29b levels in plasma, PBC and BM were measured using this assay. The PK was analyzed using WinNonlin software. Results: Excellent linearity was observed in plasma and in K562 cell lysate at a concentration range between 5-5000 pM. The intra-day and inter-day (n=6) precisions at 10, 50, 500, and 5000 pM were below 20% and the accuracy was 100±20%. Plasma and intracellular levels of O-Me PS miR-29b in PBC and BM were detectable at 24 hr after dosing with no interference in blank samples. Plasma and PBC O-Me PS miR-29b levels reached ~1.9 and 1.67 μM at 5 min, declined biexponentially, and were detectable at ~0.0013 and ~0.0025 μM at 24 h, respectively. The terminal half life was 507.4 min, AUC 115.1 min*μM and the total body clearance (CL) 0.087 mL/min/kg and the volume of distribution 8.9 mL/kg. The higher plasma level as compared to the PBC level before 7 h suggests that there was a gradient in uptake of 2-MeO PS miR-29b by blood cells. Notably, a comparable intracellular level of O-Me PS miR-29b at 0.0032 μM in BM was also detected at 24 h. Conclusion: An ultrasensitive hybridization ELISA assay for miRNAs including miR-16 and antagomiR-155 in biological matrices was developed and validated. This assay was used to study the PK study of O-Me PS miR-29b. The assay provides a valuable tool for the PK-pharmacodynamics correlation of synthetic miRNAs for their potential therapeutics.