Pharmacodynamic monitoring of BAY 43-9006 (Sorafenib) in phase I clinical trials involving solid tumor and AML/MDS patients, using flow cytometry to monitor activation of the ERK pathway in peripheral blood cells.

BACKGROUND: We previously reported a flow cytometry technique to monitor pharmacodynamic effects of the raf kinase inhibitor BAY 43-9006 based on the ability of phorbol ester (PMA) to phosphorylate extracellular-regulated kinase (ERK) in peripheral blood (Chow et al., Cytometry 2001;46:72-78). In this article, we describe its application to phase I trials of BAY 43-9006 in solid tumor and AML/MDS patients. METHODS: The previously described whole blood lysis method was used to monitor BAY 43-9006 effects on peripheral T-cells of solid tumor patients. A modified whole blood fixation protocol was developed for the AML/MDS trial, using the c-kit ligand stem cell factor (SCF) to activate ERK as an alternative to PMA, and incorporating immunophenotypic markers to identify leukemic blasts. RESULTS: At all dose levels of BAY 43-9006 used to treat solid tumor patients, ERK could be activated by PMA in peripheral T-cells and we were not able to show inhibition of raf kinase. A similar effect was seen in the lymphocytes of AML/MDS patients during treatment with BAY 43-9006. However, we found strong inhibition when ERK was activated via c-kit using SCF. Furthermore, normal donor CD34+ve stem cells were much more sensitive to BAY 43-9006 when ERK was activated by SCF, compared to PMA. CONCLUSIONS: These findings support the further development of flow cytometry applications to monitor signal transduction inhibitors during early phase clinical trials.

Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand.

In a variety of cells, the Ca2+ signalling process is mediated by the endoplasmic-reticulum-membrane-associated Ca2+ release channel, inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Being ubiquitous and present in organisms ranging from humans to Caenorhabditis elegans, InsP3R has a vital role in the control of cellular and physiological processes as diverse as cell division, cell proliferation, apoptosis, fertilization, development, behaviour, memory and learning. Mouse type I InsP3R (InsP3R1), found in high abundance in cerebellar Purkinje cells, is a polypeptide with three major functionally distinct regions: the amino-terminal InsP3-binding region, the central modulatory region and the carboxy-terminal channel region. Here we present a 2.2-A crystal structure of the InsP3-binding core of mouse InsP3R1 in complex with InsP3. The asymmetric, boomerang-like structure consists of an N-terminal beta-trefoil domain and a C-terminal alpha-helical domain containing an 'armadillo repeat'-like fold. The cleft formed by the two domains exposes a cluster of arginine and lysine residues that coordinate the three phosphoryl groups of InsP3. Putative Ca2+-binding sites are identified in two separate locations within the InsP3-binding core.