Abstract 966: Novel pharmacodynamic assays to measure glutaminase inhibition following oral administration of CB-839
Authors: MacKinnon Andy L. , Bennett Mark K. , Gross Matthew I. , Janes Julie R. , Lewis Evan R. , Rodriguez Mirna L.M. , Shwonek Peter J. , Taotao Wang , Yang Jinfu , Zhao Frances , Parlati Francesco 
Published: May 2015

Abstract
Glutaminase is a mitochondrial enzyme that plays a crucial role in tumor growth and survival. Glutaminase converts glutamine (Gln) to glutamate (Glu) fueling multiple downstream metabolic pathways required for cellular proliferation. CB-839 is a novel, selective and potent inhibitor of glutaminase that displays antitumor activity in preclinical models of several hematologic and solid tumor types. Treatment of tumor cell lines in vitro with CB-839 caused dose-dependent decreases in Glu and increases in Gln with a potency similar to the IC50 of CB-839 on recombinant glutaminase and the EC50 of CB-839 in cellular proliferation assays. Oral administration of CB-839 to mice bearing human xenograft tumors resulted in similar changes in Glu and Gln levels in the tumor. To determine if these pharmacodynamics effects of CB-839 directly correlated with inhibition of glutaminase, we developed an assay to measure glutaminase activity in tumors. Since CB-839 is a reversible inhibitor, we first developed conditions that maintain the enzyme-inhibitor complex during preparation of tumor lysates. High concentrations of KCl (150 mM) and low concentrations of K-phosphate (15 mM) in the lysis buffer, as well as maintaining the lysate at a low temperature promoted stability of the inhibited complex. Following gel filtration to remove unbound CB-839 and exchange the buffer, glutaminase activity was measured with a coupled enzyme assay. This assay was used to study the dose-dependence of glutaminase inhibition in tumors. Four hours after oral administration, a 10 mg/kg dose of CB-839 resulted in &gt;90% inhibition of tumor glutaminase and was associated with near maximal changes in tumor Gln and Glu. Moreover, CB-839 plasma concentrations of 100 nM corresponded to 50% inhibition of tumor glutaminase, while maximal inhibition occurred at plasma concentrations ≥300 nM. In xenograft studies, maximal anti-tumor efficacy was achieved with BID dosing at 200 mg/kg. This dose and schedule allowed for sustained plasma levels of CB-839 of ≥300 nM, and corresponded to sustained glutaminase inhibition in tumors. In an effort to develop a surrogate marker for inhibition of tumor glutaminase, we adapted the assay to measure glutaminase inhibition in platelets. Ex vivo treatment of human whole blood with CB-839 resulted in dose-dependent suppression of platelet glutaminase activity with an IC50 of 25 nM and &gt;85% inhibition at concentrations at or above 300 nM. Furthermore, glutaminase activity in platelets isolated from mice treated with CB-839 showed dose-dependent inhibition that correlated with inhibition of tumor glutaminase. Thus, platelet glutaminase activity may represent a surrogate for monitoring inhibition of glutaminase in human tumors. These assays will be applied to the clinical study of CB-839 to monitor pharmacodynamic responses and to ensure maximal inhibition of glutaminase in patients undergoing therapy.
Citation Format: Andy L. MacKinnon, Mark K. Bennett, Matthew I. Gross, Julie R. Janes, Evan R. Lewis, Mirna L.M. Rodriguez, Peter J. Shwonek, Wang Taotao, Jinfu Yang, Frances Zhao, Francesco Parlati. Novel pharmacodynamic assays to measure glutaminase inhibition following oral administration of CB-839. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 966. doi:10.1158/1538-7445.AM2014-966