Validation of in vivo pharmacodynamic activity of a novel PDGF receptor tyrosine kinase inhibitor using immunohistochemistry and quantitative image analysis
Validation of in vivo pharmacodynamic activity of a novel PDGF receptor tyrosine kinase inhibitor using immunohistochemistry and quantitative image analysis
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DOI：
10.1158/1535-7163.mct-05-0004
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发表时间：
2005-08-01
影响因子：
5.7
通讯作者：
Johnson, DL
中科院分区：
文献类型：
--
作者：
D'Andrea, MR;Mei, JM;Johnson, DL
随着药物发现中针对特定分子靶点的药物的出现，在体内展示化合物对预期生物分子的细胞影响已变得势在必行。本研究的目的是确定我们是否可以开发一种方法来验证一种化合物的体内效应。因此，我们研究了相对选择性的血小板衍生生长因子受体酪氨酸激酶抑制剂JNJ-10198409在裸鼠结肠癌裸鼠移植瘤模型中的体内药效学活性。我们开发了一种新的方法来定量该抑制物在体内的抗PDGF-RTK活性，方法是通过测定磷脂酶C-伽马1(PLC-伽马1)的磷酸化状态，磷脂酶C-伽马1是PDGF-RTK信号级联中的关键下游细胞分子。我们使用了两种抗体，一种是针对全部(磷酸化和非磷酸化形式)PLC Gamma 1的抗体(PAN-PLC Gamma 1)，另一种是针对磷酸化形式的PLC Gamma 1(ph-PLC Gamma 1)的抗体，用免疫组织化学方法检测了它们在肿瘤组织中的表达。然后利用计算机辅助图像分析直接比较从赋形剂和JNJ-10198409治疗的荷瘤小鼠获得的肿瘤连续切片(5um M)中ph-PLC-Gamma 1与PAN-PLC Gamma 1免疫标记强度的比值。我们的数据显示，在四个治疗组(赋形剂、25、50和100 mg/kg b.i.d)之间，ph-PLC/PAN-PLC比率具有统计学上的剂量依赖性差异。这些结果证实了该化合物在体内抑制肿瘤组织中PDGF-RTK下游信号的能力。除了将这种体内验证方法具体应用于那些使用PLC Gamma作为下游信号伙伴的靶点外，这些方法还可能使其他药物发现靶点受益。
With the advent of agents directed against specific molecular targets in drug discovery, it has become imperative to show a compound's cellular impact on the intended biomolecule in vivo. The objective of the present study was to determine if we could develop an assay to validate the in vivo effects of a compound. Hence, we investigated the in vivo pharmacodynamic activity of JNJ-10198409, a relatively selective inhibitor of platelet-derived growth factor receptor tyrosine kinase (PDGFRTK), in tumor tissues after administering the compound orally in a nude mouse xenograft model of human LoVo colon cancer. We developed a novel assay to quantify the in vivo anti-PDGF-RTK activity of the inhibitor in tumor tissue by determining the phosphorylation status of phospholipase C gamma 1 (PLC gamma 1), a key downstream cellular molecule in the PDGF-RTK signaling cascade. We used two antibodies, one specific for the total (phosphorylated and unphosphorylated forms) PLC gamma 1 (pan-PLC gamma 1) and the other, specific for phosphorylated form of PLC gamma 1 (ph-PLC gamma 1) to immunohistochemically detect their expression in tumor tissues. Computer-assisted image analysis was then used to directly compare the ratio of ph-PLC gamma 1 to pan-PLC gamma 1 immunolabeling intensities in serial sections (5 mu m) of tumors obtained from vehicle- and JNJ-10198409-treated tumor-bearing mice. Our data showed statistically significant, dose-dependent differences in the ph-PLC/pan-PLC ratio among the four treatment groups (vehicle, 25, 50, and 100 mg/kg b.i.d.). These results confirmed this compound's ability to suppress PDGF-RTK downstream signaling in tumor tissues in vivo. In addition to this specific application of this in vivo validation approach to those targets that use PLC gamma as a downstream signaling partner, these methods may also benefit other drug discovery targets.