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Drug Discovery, Johnson & Johnson Pharmaceutical Research and Development, L.L.C., Spring House, Pennsylvania

Requests for reprints: Michael R. D'Andrea, 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. Phone: 215-628-5619; Fax: 215-540-4887. E-mail: mdandrea{at}prdus.jnj.com

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 C1 (PLC1), a key downstream cellular molecule in the PDGF-RTK signaling cascade. We used two antibodies, one specific for the total (phosphorylated and unphosphorylated forms) PLC1 (pan-PLC1) and the other, specific for phosphorylated form of PLC1 (ph-PLC1) to immunohistochemically detect their expression in tumor tissues. Computer-assisted image analysis was then used to directly compare the ratio of ph-PLC1 to pan-PLC1 immunolabeling intensities in serial sections (5 µm) 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 as a downstream signaling partner, these methods may also benefit other drug discovery targets.

¹ Manuscript in preparation.

Received 1/ 5/05; revised 6/ 8/05; accepted 6/14/05.