Dai Guowei, Chan Kenneth K, Liu Shujun, Hoyt Dale, Whitman Susan, Klisovic Marko, Shen Tiansheng, Caligiuri Michael A, Byrd John, Grever Michael, Marcucci Guido
Division of Pharmaceutics, College of Medicine and Public Health, Ohio State University, Columbus, Ohio 43210, USA.
Clin Cancer Res. 2005 Apr 15;11(8):2998-3008. doi: 10.1158/1078-0432.CCR-04-1505.
Down-regulation of Bcl-2 by the antisense G3139, currently under clinical evaluations, could restore chemosensitivity in otherwise resistant malignant cells. To date, the mechanism of intracellular accumulation of G3139 following in vivo administration remains to be elucidated. This study aimed to assess whether detectable intracellular concentrations of G3139 are achievable in vivo and how these relate to Bcl-2 down-regulation.
Cellular uptake of G3139 was studied in leukemia myeloid cell lines and blasts collected from treated patients using a newly developed, novel, and highly sensitive ELISA-based assay. Real-time reverse transcription-PCR was used to quantify Bcl-2 mRNA changes in treated cells.
The assay was fully validated and showed a limit of quantification of 50 pmol/L. When exposed to 0.33 to 10 mumol/L G3139, K562 cells exhibited intracellular concentrations in the range of 2.1 to 11.4 pmol/mg protein. When G3139 was delivered with cationic lipids, a 10- to 25-fold increase of the intracellular concentrations was observed. There was an accumulation of G3139 in the nuclei, and the ratio of nucleus to cytoplasm was increased 7-fold by cationic lipids. Intracellular concentrations of G3139 were correlated with Bcl-2 mRNA down-regulation. Robust intracellular concentrations of G3139 were achieved in vivo in bone marrow (range, 3.4-40.6 pmol/mg protein) and peripheral blood mononuclear cells (range, 0.47-19.4 pmol/mg protein) from acute myeloid leukemia patients treated with G3139.
This is the first evidence that measurable intracellular levels of G3139 are achievable in vivo in acute myeloid leukemia patients and that Bcl-2 down-regulation is likely to depend on the achievable intracellular concentrations rather than on plasma concentrations.
目前正在进行临床评估的反义寡核苷酸G3139可下调Bcl-2，从而恢复原本耐药的恶性细胞的化疗敏感性。迄今为止，体内给药后G3139在细胞内蓄积的机制仍有待阐明。本研究旨在评估体内是否能达到可检测的细胞内G3139浓度，以及这些浓度与Bcl-2下调之间的关系。
采用新开发的、新颖且高度灵敏的基于酶联免疫吸附测定（ELISA）的方法，研究白血病髓系细胞系以及从接受治疗的患者采集的原始细胞对G3139的细胞摄取情况。使用实时逆转录-聚合酶链反应（RT-PCR）定量处理后细胞中Bcl-2 mRNA的变化。
该检测方法经过全面验证，定量限为50 pmol/L。当暴露于0.33至10 μmol/L的G3139时，K562细胞内浓度范围为2.1至11. pmol/mg蛋白质。当G3139与阳离子脂质一起递送时，细胞内浓度增加了10至25倍。G3139在细胞核中蓄积，阳离子脂质使细胞核与细胞质的比例增加了7倍。G3139的细胞内浓度与Bcl-2 mRNA下调相关。在接受G3139治疗的急性髓系白血病患者的骨髓（范围为3.4 - 40.6 pmol/mg蛋白质）和外周血单个核细胞（范围为0.47 - 19.4 pmol/mg蛋白质）中，体内实现了较高的G3139细胞内浓度。
这是首个证据表明，在急性髓系白血病患者体内可实现可测量的细胞内G3139水平，且Bcl-2下调可能取决于可达到的细胞内浓度而非血浆浓度。
