Development and Validation of a Drug Activity Biomarker that Shows Target Inhibition in Cancer Patients Receiving Enzastaurin, a Novel Protein Kinase C-β Inhibitor

Abstract

Purpose: To evaluate the effects of the novel protein kinase C (PKC) inhibitor enzastaurin on intracellular phosphoprotein signaling using flow cytometry and to use this approach to measure enzastaurin effects on surrogate target cells taken from cancer patients that were orally dosed with this agent. Experimental Design: The activity of PKC was assayed in intact cells using a modification of published techniques.The U937 cell line and peripheral blood mononuclear cells were stimulated with phorbol ester, fixed, permeabilized, and reacted with an antibody specific for the phosphorylated forms of PKC substrates. The processed samples were quantitatively analyzed using flow cytometry. The assay was validated for selectivity, sensitivity, and reproducibility. Finally, blood was obtained from volunteer cancer patients before and after receiving once daily oral doses of enzastaurin. These samples were stimulated ex vivo with phorbol ester and were assayed for PKC activity using this approach. Results: Assay of U937 cells confirmed the selectivity of the antibody reagent and enzastaurin for PKC. Multiparametric analysis of peripheral blood mononuclear cells showed monocytes to be the preferred surrogate target cell. Day-to-day PKC activity in normal donors was reproducible. Initial results showed that five of six cancer patients had decreased PKC activity following enzastaurin administration. In a following study, a group of nine patients displayed a significant decrease in PKC activity after receiving once daily oral doses of enzastaurin. Conclusion: An inhibition of surrogate target cell PKC activity was observed both in vitro and ex vivo after exposure to the novel kinase inhibitor, enzastaurin.

Protein Kinase C as a Therapeutic Target

“…Monocytes collected from patients treated with enzastaurin showed decreased PKC activity after ex vivo stimulation, with phorbol ester providing a potential biomarker in a surrogate cell (86). Phase II studies have been conducted in patients with relapsed diffuse large B cell lymphoma and in patients with recurrent high-grade gliomas.…”

Enzastaurin shows preclinical antitumor activity against human transitional cell carcinoma and enhances the activity of gemcitabine

“…Enzastaurin is an orally administered serine/threonine kinase inhibitor that suppresses the protein kinase C (PKC)-β, phosphatidylinositol 3-kinase/AKT, GSK-3 β, and vascular endothelial growth factor signaling pathways (5,6). Enzastaurin is feasible as a single agent and in combination with cisplatin plus gemcitabine in early phase I clinical trials, and is active in phase II trials of lymphomas (7)(8)(9)(10).…”

A platelet biomarker for assessing phosphoinositide 3-kinase inhibition during cancer chemotherapy

“…In the clinical setting, blood is an advantageous sample source for biomarker assays due to the ease of obtaining, storing, and transporting the specimen. Blood specimens can provide a disease target tissue (leukemia cells), or in the case of solid tumors, the normal blood components can provide surrogate targets for pharmacologic investigation (15). With the development of less toxic therapies, drug activity biomarkers have become increasingly important for estimating efficacious doses of an experimental agent.…”