Purpose
To develop and validate an ultrasensitive and specific hybridization-based enzyme-linked immunosorbent assay method for quantification of two phosphorothioate oligonucleotides (PS ODNs) (G3139 and GTI-2040) in biological fluids.
Methods
This assay was based on hybridization of analytes to the biotin-labeled capture ODNs followed by ligation with digoxigenin-labeled detection ODN. The bound duplex was then detected by anti-digoxigenin-alkaline phosphatase using Attophos® (Promega, Madison, WI, USA) as substrate. S1 nuclease and major factors such as the hybridization temperature, concentration of capture probe, and the use of detergent were evaluated toward assay sensitivity, selectivity, and accuracy.
Results
The method is selective to the parent drugs with minimal cross-reactivity (<6%) with 3′-end deletion oligomers for both G3139 and GTI-2040. A linear range of 0.05 to 10 nM (r2 > 0.99) was observed for GTI-2040 in a variety of biological matrices. For both G3139 and GTI-2040, the within-day precision and accuracy values were found to be <20% and 90–110%, respectively; the between-day precision and accuracy were determined to be <20% and 90–120%. Addition of S1 nuclease combined with washing step greatly improved the assay linearity and selectivity. The utility of this assay was demonstrated by simultaneous determination of GTI-2040 in plasma and its intracellular levels in treated acute myeloid leukemia patients.
Conclusions
The validated hybridization enzyme-linked immunosorbent assay method is specific for quantitation of PS ODNs in biological samples to picomolar level. This method provides a powerful technique to evaluate plasma pharmacokinetics and intracellular uptake of PS ODNs in patients and shows its utility in clinical evaluations.
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Abbreviations
- AML:
-
acute myeloid leukemia
- AP:
-
alkaline phosphatase
- AS ODN:
-
antisense oligonucleotide
- AUC:
-
area under the curve
- BM:
-
bone marrow
- CL:
-
total body clearance
- C ss :
-
steady state concentration
- CV:
-
coefficient of variation
- Dig:
-
digoxigenin
- ELISA:
-
enzyme-linked immunosorbent assay
- LLOQ:
-
lower limit of quantification
- LOD:
-
limit of detection
- mRNA:
-
messenger ribonucleic acid
- PBMC:
-
peripheral blood mononuclear cell
- PD:
-
pharmacodynamics
- PK:
-
pharmacokinetics
- PS ODN:
-
phosphorothioate oligonucleotide
- RBC:
-
red blood cells
- RNase H:
-
ribonuclease H
- RNR:
-
ribonucleotide reductase
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Acknowledgments
We acknowledge the support by NIH R21CA105879 and UO1 CA 76576.
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Wei, X., Dai, G., Marcucci, G. et al. A Specific Picomolar Hybridization-Based ELISA Assay for the Determination of Phosphorothioate Oligonucleotides in Plasma and Cellular Matrices. Pharm Res 23, 1251–1264 (2006). https://doi.org/10.1007/s11095-006-0082-3
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DOI: https://doi.org/10.1007/s11095-006-0082-3