Abstract 3620: Tumor targeted delivery of glutamine antagonist: Use of CES1-/- mice
Authors: Rana Rais, Jesse Alt, Ranjeet Dash, Lukáš Tenora, Pavel Majer, Barbara S. Slusher
Journal: Cancer Research (2019)

Abstract 6-diazo-5-oxo-L-norleucine (DON), a potent glutamine antagonist, broadly blocks glutamine utilizing reactions critical for the synthesis of nucleic acids, amino acids, proteins and the generation of alpha-ketoglutarate for energy metabolism. DON has shown robust efficacy in multiple preclinical cancer models and exploratory clinical studies. Although promising, development of DON was halted due to its dose-limiting gastrointestinal (GI)-toxicities, as the GI system is highly dependent on glutamine utilization. Given DON’s promising efficacy, we developed novel tumor cell-targeted glutamine antagonists intended to circulate intact as inert prodrug/s in plasma and be preferentially biotransformed to DON in tumor cells. Our prodrug strategy of utilizing elevated protease activities in cancer tissues (e.g. Histone deacetylase, Cathepsin-L) led to the discovery of a highly innovative prodrug pharmacophore. Using a well-defined screening paradigm, we discovered compound 6, (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido) hexanamido)-6-diazo-5-oxohexanoate), that showed stability in plasma, liver and intestinal homogenates, yet was readily cleaved to DON in P493B human tumor cells. When directly compared to DON, compound 6, exhibited a 55-fold enhanced P493B cell-to-plasma ratio. In a time-dependent study, compound 6 showed sustained DON delivery to P493B cells while maintaining minimal release in human plasma. Moreover, in a cell proliferation assay, compound 6 showed dose-dependent inhibition of P493B cell growth. One challenging aspect of prodrug development is the selection of appropriate animal model, as it can be confounded by interspecies variation in metabolism, specifically in the varying levels of the carboxylesterase enzyme, CES1, which is highly abundant in rodent plasma but not present in human plasma. We hypothesized that CES1-/- mice would recapitulate human metabolism and serve as a suitable model for our prodrug containing ester promoiety on its carboxylate. Using plasma from CES1-/- mice, wild-type mice and human, we confirmed that compound 6 exhibited similar stability in CES1-/- mice and human plasma but not in wild-type mice plasma. We then performed pharmacokinetic evaluation in C57BL/6 CES1-/- mice bearing flank murine EL4 tumors. Following subcutaneous dosing (1mg/kg DON equivalent), compound 6 exhibited excellent pharmacokinetics with a ~5-fold higher DON tumor exposures (AUC= 5.1 nmol/g*h) versus plasma (1.1 nmol/ml*h) and a 11-fold higher tumor exposures versus GI-tissues (toxicity site; AUC = 0.45 nmol/ml*h). These studies describe discovery of a tumor targeted glutamine antagonist. In addition, we introduce a murine model, that recapitulates human metabolism and can be broadly utilized in prodrug development. Future studies will investigate the dose dependent efficacy and safety of compound 6 in tumor bearing C57BL/6 CES1-/- mice. Citation Format: Rana Rais, Jesse Alt, Ranjeet Dash, Lukáš Tenora, Pavel Majer, Barbara S. Slusher. Tumor targeted delivery of glutamine antagonist: Use of CES1-/- mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3620.