We're Not "DON" Yet: Optimal Dosing and Prodrug Delivery of 6-Diazo-5-oxo-L-norleucine

Abstract

The broadly active glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) has been studied for 60 years as a potential anticancer therapeutic. Clinical studies of DON in the 1950s using low daily doses suggested antitumor activity, but later phase I and II trials of DON given intermittently at high doses were hampered by dose-limiting nausea and vomiting. Further clinical development of DON was abandoned. Recently, the recognition that multiple tumor types are glutamine-dependent has renewed interest in metabolic inhibitors such as DON. Here, we describe the prior experience with DON in humans. Evaluation of past studies suggests that the major impediments to successful clinical use included unacceptable gastrointestinal (GI) toxicities, inappropriate dosing schedules for a metabolic inhibitor, and lack of targeted patient selection. To circumvent GI toxicity, prodrug strategies for DON have been developed to enhance delivery of active compound to tumor tissues, including the CNS. When these prodrugs are administered in a low daily dosing regimen, appropriate for metabolic inhibition, they are robustly effective without significant toxicity. Patients whose tumors have genetic, metabolic, or imaging biomarker evidence of glutamine dependence should be prioritized as candidates for future clinical evaluations of novel DON prodrugs, given either as monotherapy or in rationally directed pharmacologic combinations. Mol Cancer Ther; 17(9); 1824-32. ©2018 AACR.

Figure 1:. Cellular Glutamine Utilization Targeted by DON.

Top: Illustration depicting major glutamine utilizing pathways in mammalian cells with target enzymes (pink abbreviations) known to be inhibited by DON. Bottom: List of known pathways and enzymes affected by DON with established Ki values where available.

Figure 2:. DON prodrugs enhance DON tissue-to-plasma ratios and possess potent antitumor activity without overt toxicities.

(A) Structures of DON Prodrugs. (B) Prodrug 1 preferentially delivers DON to the CNS. DON and Prodrug 1 were dosed by IV infusion (1.6 mg/kg equivalent doses for 1h) in swine and DON was quantified in plasma, CSF and brain samples. Prodrug 1 delivered DON preferentially to CSF (i) and brain (ii) resulting in 15-fold enhanced CSF/plasma ratio and 9 fold enhanced brain/plasma ratio when compared directly with equimolar doses of DON. Adapted with permission from reference (58). Copyright 2017 American Chemical Society. (C) Prodrug 2 preferentially delivers DON to P493B tumor cells. In head-to-head comparison, Prodrug 2 shows a 90-fold enhanced tumor cell/plasma ratio versus DON. (D) Prodrug 2 does not release significant DON in gastrointestinal tissue. Prodrug 2 was incubated with pig gastrointestinal homogenate for 60 minutes. At 60 minutes, DON liberation from Prodrug 2 was limited to 20%. (E) Daily dosing of Prodrug 3 eliminates EL4 engrafted tumors without weight loss or overt toxicity. 8–10 week old male C57BL/6 mice (6–7 per treatment group) were injected subcutaneously into the right flank with 1×10⁶ EL4 cells on day 0, and after five days of growth the mice had measurable tumors. On day 6 after implantation, treatment was initiated with either vehicle or DON Prodrug (1 mg/kg p.o. daily × 5 days followed by 0.3 mg/kg, p.o.). Tumors were measured every other day with calipers and volumes calculated by the formula V=(LxW²)/2. DON prodrug eliminated tumor in all mice, while mice treated with vehicle succumbed to tumors by day 13.