Mechanism of Action of G3139

描述（由申请人提供）：最近使用G3139（又名Oblimersen）的临床前和II期临床试验数据令人兴奋，G3139是一种18mer I反义硫代寡脱氧核苷酸，靶向bcl-2 mRNA的前六个密码子，表明这种新型药物与细胞毒性化疗联合治疗相对较多的人类肿瘤可能具有临床活性。然而，对于这种药物的确切作用机制，仍然存在重要的问题。我们和其他人已经确定了G3139的四种不同的可能的作用机制：1)下调bcl-2，并假定随后特异性增加化学敏感性；2)寡核苷酸的非特异性作用，包括不相关的裂解，导致与机制1协同作用；3) CpG基序调节的免疫效应机制刺激，可能涉及toll样受体；4)局部（在肿瘤水平）CpG调节的活性氧（ROS）的产生导致细胞生长速率的降低。为了证明G3139在体外和体内抗肿瘤作用的机制，我们设计了四个具体目标：具体目标1：我们将确定G3139及其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。我们将加入小分子清除ROS和H202，在外媒中加入过氧化氢酶阻断H202对细胞生长的影响，用腺病毒- mnsod载体感染细胞

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.