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Pharmaceutical Research

, Volume 23, Issue 6, pp 1251–1264 | Cite as

A Specific Picomolar Hybridization-Based ELISA Assay for the Determination of Phosphorothioate Oligonucleotides in Plasma and Cellular Matrices

  • Xiaohui Wei
  • Guowei Dai
  • Guido Marcucci
  • Zhongfa Liu
  • Dale Hoyt
  • William Blum
  • Kenneth K. Chan
Research Paper

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.

Key Words

hybridization–ligation ELISA intracellular drug level pharmacokinetics phosphorothioate oligonucleotides 

Abbreviations

AML

acute myeloid leukemia

AP

alkaline phosphatase

AS ODN

antisense oligonucleotide

AUC

area under the curve

BM

bone marrow

CL

total body clearance

Css

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

Notes

Acknowledgments

We acknowledge the support by NIH R21CA105879 and UO1 CA 76576.

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Copyright information

© Springer Science + Business Media, Inc. 2006

Authors and Affiliations

  • Xiaohui Wei
    • 1
  • Guowei Dai
    • 1
  • Guido Marcucci
    • 2
    • 3
  • Zhongfa Liu
    • 1
  • Dale Hoyt
    • 4
  • William Blum
    • 3
  • Kenneth K. Chan
    • 1
    • 2
  1. 1.Division of Pharmaceutics, College of PharmacyThe Ohio State UniversityColumbusUSA
  2. 2.The Comprehensive Cancer Center, Rm 308 OSU CCCThe Ohio State UniversityColumbusUSA
  3. 3.Division of Hematology-Oncology, College of Medicine and Public HealthThe Ohio State UniversityColumbusUSA
  4. 4.Division of Pharmacology, College of PharmacyThe Ohio State UniversityColumbusUSA

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