Abstract 2540: Cytotoxicity, pharmacokinetics and metabolism of 10074-G5, a novel small-molecule inhibitor of c-Myc
Authors: Clausen Dana , Guo Jianxia , Beumer Jan H. , Parise Robert A. , Bai Lihua , Egorin Merrill J. , Lazo John S. , Prochownik Edward V. , Eiseman Julie L. 
Published: February 2011

Abstract
Introduction
The transcription factor c-Myc plays an important role in cellular proliferation, differentiation, apoptosis and cell cycle progression; is overexpressed in numerous cancer types; and is associated with cancer aggressiveness and poor prognosis. In order to be transcriptionally active, c-Myc must heterodimerize via its bHLH-ZIP domain with its obligatory partner Max. 10074-G5 is a small molecule that prevents c-Myc/Max heterodimerization, thereby inhibiting c-Myc function. We have characterized the ability of 10074-G5 to inhibit the in vitro growth of 2 c-Myc-overexpressing human cell lines: Daudi, a Burkitt's lymphoma; and HL-60, a myelocytic leukemia. We also analyzed the effect of 10074-G5 on c-Myc/Max heterodimer formation and total c-Myc protein expression in Daudi cells and evaluated the pharmacokinetics and metabolism of 10074-G5 in mice bearing Daudi xenografts.
Methods
Inhibition of Daudi and HL-60 cell growth by 10074-G5 was assessed by MTT assay after 72 h of incubation. Accumulation of 10074-G5 by Daudi cells incubated with 10 μM 10074-G5 was measured by HPLC. c-Myc/Max co-immunoprecipitations and Western blots were performed on whole cell lysate from control and 10 μM 10074-G5-treated Daudi cells. For pharmacokinetic and metabolic studies, C.B-17 SCID mice bearing Daudi xenografts were treated with 20 mg/kg 10074-G5 iv. Plasma and tissues were collected at 12 specified time points between 5 and 1440 min. Urine was collected at 360 and 1440 min. Concentrations of 10074-G5 were determined by HPLC-UV, and metabolites were identified by LC-MS/MS.
Results
10074-G5 inhibited in vitro growth of Daudi cells (IC50 15.6 µM) and HL-60 cells (IC50 13.5 µM). Daudi cells accumulated 10074-G5 approximately 50-fold, and peak intracellular concentrations of &gt;20 nmoles/108 cells were observed between 4 and 8 h of incubation. After exposure to 10 µM 10074-G5, c-Myc/Max heterodimer formation was inhibited at 4 h, and c-Myc total protein expression was decreased by 70% after 24 h. In mice, 10074-G5 was non-toxic at the highest soluble dose of 20 mg/kg iv. 10074-G5 had a peak plasma concentration of 58 µM, a plasma half-life of 37 min, and concentrations in plasma were below the limit of detection beyond 240 min. Structural elucidation of 19 metabolites by LC-MS/MS indicated that hydroxylation and glucuronidation were the major metabolic pathways of 10074-G5. Concentrations of 10074-G5 in the tumor peaked at 5 min and were 5.6 µM, which was 10-fold lower than concomitant plasma concentrations. After 120 min, tumor concentrations of 10074-G5 were below the limit of detection.
Conclusion
10074-G5 is rapidly metabolized in mice, and, based on in-vitro data, tumor concentrations of 10074-G5 at the currently administered dose are too low to inhibit c-Myc/Max dimerization. Evaluation of more metabolically stable and soluble analogues is warranted.
(Support: P01-CA078039 and P30-CA47904)
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2540.