Mechanism of Action of G3139

描述(由申请人提供)：采用G3139的最新令人兴奋的临床前和第二阶段临床试验数据(也称为。Oblimersen)，一个针对bcl2mRNA前6个密码子的18聚I反义硫代寡核苷酸，表明这种新型药物与细胞毒化疗联合治疗相对较大数量的人类肿瘤可能具有临床活性。然而，关于该制剂的确切作用机制仍存在重大问题。我们和其他人已经确定了G3139的四种不同的可能作用机制：1)bcl2下调，推测随后化疗敏感性特异性增加；2)寡核苷酸的非特异性作用，包括无关的切割，导致与机制1的协同作用；3)CpG基序调节的免疫效应机制的刺激，可能涉及Toll样受体，以及4)局部(在肿瘤水平)CpG调节的活性氧物种(ROS)的产生，导致细胞生长速度减慢。为了证明G3139在体外和体内的抗肿瘤作用的机制，我们设计了四个特定的目标：特定的目标1：我们将确定G3139和相关寡核苷酸在前列腺和膀胱细胞中产生的ROS和H202如何影响这些细胞的生长和活力。我们将添加小分子来清除ROS和H202，在外部培养基中添加过氧化氢酶来阻断H202对细胞生长的影响，并用腺病毒-MnSOD载体感染细胞 减少ROS的生产。我们还将确定H202的细胞内浓度，并确定这些寡核苷酸以bcl2依赖和独立的方式诱导脂质过氧化的能力。我们还将在G3139存在的情况下检测线粒体功能(例如，线粒体潜力、ATP产生、氧气消耗)。具体目标2：我们将在G3139的3‘和5’末端使用新型的2‘-O，4’-C-亚甲基连接的双环核糖核苷(LNAs)来增加其靶标的寡聚稳定性和亲和力。除了与G3139相比能够下调bcl-2的表达外，还将确定最佳的Gapmer。具体目的3：我们将使用SCID小鼠的PC3细胞异种移植来评价免疫刺激下调bcl2表达和局部产生活性氧在G3139及其LNA GAP-mer同系物抗肿瘤作用中的作用。最后，在特定的目标4中，我们将对G3139和相关的寡核苷酸处理的前列腺和膀胱癌细胞进行Affymetrix基因芯片分析，以特异性地确定哪些基因受到G3139处理的影响。

DESCRIPTION (provided by applicant): Recent exciting pre-clinical and phase II clinical trial data employing G3139 (a.k.a. Oblimersen), an 18mer I antisense phosphorothioate oligodeoxynucleotide targeted to the first six codons of the bcl-2 mRNA, indicates that this novel agent may be clinically active in combination with cytotoxic chemotherapy in the treatment of a relatively large number of human tumors. However, significant questions remain as to the precise mechanism of action of this agent. We and others have ascertained four distinct possible mechanisms of action of G3139: 1) Downregulation of bcl-2 with postulated subsequent specific increase in chemosensitivity; 2) Non-specific effects of the oligonucleotide, including irrelevant cleavage, leading to synergy with mechanism 1; 3) CpG motif-modulated stimulation of immune effector mechanisms, possibly involving Toll-like receptors, and 4) Local (at the level of the tumor) CpG-modulated production of reactive oxygen species (ROS) leading a diminution in the rate of cell growth. In order to demonstrate which mechanisms are responsible for the antitumor effects of G3139 both in vitro and in vivo, we have devised four Specific Aims: Specific Aim 1: We will determine how the production of ROS and H202 by G3139 and related oligos in prostate and bladder cells serves to affect the growth and viability of these cells. We will add small molecules to scavenge ROS and H202, add catalase to the external media to block the effects of H202 on cell growth, and infect cells with an adenoviral-MnSOD vector to reduce ROS production. We will also determine the intracellular concentration of H202, and determine the ability of these oligos to induce lipid-peroxidation in a bcl-2 dependent and independent manner. We will also examine mitochondrial function in the presence of G3139 (e.g., mitochondrial potential, ATP production, oxygen consumption). Specific Aim 2: We will employ novel 2'-O,4'-C-methylene-linked bicyclic ribonucleosides (LNAs) at the 3' and 5' termini of G3139 to increase oligo stability and affinity for its target. The optimal gapmer will be determined, in addition to its ability to downregulate expression of bcl-2 compared to G3139. Specific Aim 3: We will use PC3 cell xenografts in SCID mice to evaluate the roles of immunostimulation downregulation of bcl-2 expression, and the local production of reactive oxygen species in the antitumor effect of G3139 and its LNA gap-mer homolog. Finally, in Specific Aim 4, we will perform Affymetrix gene-chip analysis of G3139 and related oligo-treated prostate and bladder carcinoma cells in order to specifically determine which genes are affected by G3139 treatment.