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.
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 leukemiapatients. 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.
Authors: F I Raynaud; R M Orr; P M Goddard; H A Lacey; H Lancashire; I R Judson; T Beck; B Bryan; F E Cotter Journal: J Pharmacol Exp Ther Date: 1997-04 Impact factor: 4.030
Authors: Guido Marcucci; Wendy Stock; Guowei Dai; Rebecca B Klisovic; Shujun Liu; Marko I Klisovic; William Blum; Cheryl Kefauver; Dorie A Sher; Margaret Green; Mollie Moran; Kati Maharry; Steven Novick; Clara D Bloomfield; James A Zwiebel; Richard A Larson; Michael R Grever; Kenneth K Chan; John C Byrd Journal: J Clin Oncol Date: 2005-04-11 Impact factor: 44.544
Authors: P A Cossum; H Sasmor; D Dellinger; L Truong; L Cummins; S R Owens; P M Markham; J P Shea; S Crooke Journal: J Pharmacol Exp Ther Date: 1993-12 Impact factor: 4.030
Authors: R Zhang; J Yan; H K Shahinian; H Shahinian; G Amin; Z Lu; T Liu; M S Saag; Z Jiang; J Temsamani; R R Martin Journal: Clin Pharmacol Ther Date: 1995-07 Impact factor: 6.875
Authors: Rebecca B Klisovic; William Blum; Zhongfa Liu; Zhiliang Xie; Cheryl Kefauver; Lenguyen Huynh; James A Zwiebel; Steven M Devine; John C Byrd; Michael R Grever; Kenneth K Chan; Guido Marcucci Journal: Leuk Lymphoma Date: 2013-11-01
Authors: Mark H Kirschbaum; Paul Frankel; Timothy W Synold; Zhiliang Xie; Yun Yen; Leslie Popplewell; Robert Chen; Omar Aljitawi; Joseph M Tuscano; Kenneth K Chan; Edward M Newman Journal: Leuk Lymphoma Date: 2016-02-19
Authors: Natasha B Leighl; Scott A Laurie; Xueyu E Chen; Peter Ellis; Frances A Shepherd; Jennifer J Knox; Glenwood Goss; Ronald L Burkes; Gregory R Pond; Christopher Dick; Yun Yen; James A Zwiebel; Malcolm J Moore Journal: J Thorac Oncol Date: 2009-09 Impact factor: 15.609
Authors: Rebecca B Klisovic; William Blum; Xiaohui Wei; Shujun Liu; Zhongfa Liu; Zhiliang Xie; Tamara Vukosavljevic; Cheryl Kefauver; Lenguyen Huynh; Jiuxia Pang; James A Zwiebel; Steven Devine; John C Byrd; Michael R Grever; Kenneth Chan; Guido Marcucci Journal: Clin Cancer Res Date: 2008-06-15 Impact factor: 12.531