title: Validation of in vivo pharmacodynamic activity of a novel PDGF receptor tyrosine kinase inhibitor using immunohistochemistry and quantitative image analysis.
abstract: 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.
authors.affiliation: Drug Discovery, D404, Johnson & Johnson Pharmaceutical Research and Development, L.L.C., P.O. Box 776, Welsh and McKean Roads, Spring House, PA 19477-0776, USA. mdandrea@prdus.jnj.com
authors.forename: Michael R
authors.initials: MR
authors.lastname: D'Andrea
authors.forename: Jay M
authors.initials: JM
authors.lastname: Mei
authors.forename: Robert W
authors.initials: RW
authors.lastname: Tuman
authors.forename: Robert A
authors.initials: RA
authors.lastname: Galemmo
authors.forename: Dana L
authors.initials: DL
authors.lastname: Johnson
mesh_terms: D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000276:immunology; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D003110:Colonic Neoplasms / Q000201:enzymology; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D007189:Indans / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D051966:Phospholipase C gamma; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011720:Pyrazoles / Q000737:chemistry / Q000494:pharmacology*; D017479:Receptors, Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors*; D010738:Type C Phospholipases / Q000032:analysis / Q000276:immunology; D023041:Xenograft Model Antitumor Assays
chemical_list: C505208:(6,7-dimethoxy-2,4-dihydroindeno(1,2-c)pyrazol-3-yl)(3-fluorophenyl)amine; D045163:Antibodies, Phospho-Specific; D000970:Antineoplastic Agents; D007189:Indans; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D017479:Receptors, Platelet-Derived Growth Factor; D010738:Type C Phospholipases; D051966:Phospholipase C gamma