Abstract 966: Novel pharmacodynamic assays to measure glutaminase inhibition following oral administration of CB-839

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 >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 >85% inhibition at concentrations at or above 300 nM. Furthermore, glutaminase activity in