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 (
PDGF-RTK), 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 Cgamma1 (PLCgamma1), a key downstream cellular molecule in the
PDGF-RTK signaling cascade. We used two
antibodies, one specific for the total (phosphorylated and unphosphorylated forms) PLCgamma1 (pan-PLCgamma1) and the other, specific for phosphorylated form of PLCgamma1 (ph-PLCgamma1) to immunohistochemically detect their expression in
tumor tissues. Computer-assisted image analysis was then used to directly compare the ratio of ph-PLCgamma1 to pan-PLCgamma1 immunolabeling intensities in serial sections (5 mum) 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 PLCgamma as a downstream signaling partner, these methods may also benefit other
drug discovery targets.