Tumor-Targeted Delivery of 6-Diazo-5-oxo-l-norleucine (DON) Using Substituted Acetylated Lysine Prodrugs

Abstract

6-Diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist with robust anticancer efficacy; however, its therapeutic potential was hampered by its biodistribution and toxicity to normal tissues, specifically gastrointestinal (GI) tissues. To circumvent DON’s toxicity, we synthesized a series of tumor-targeted DON prodrugs designed to circulate inert in plasma and preferentially activate over DON in tumor. Our best prodrug 6 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)­hexanamido)-6-diazo-5-oxohexanoate) showed stability in plasma, liver, and intestinal homogenates yet was readily cleaved to DON in P493B lymphoma cells, exhibiting a 55-fold enhanced tumor cell-to-plasma ratio versus that of DON and resulting in a dose-dependent inhibition of cell proliferation. Using carboxylesterase 1 knockout mice that were shown to mimic human prodrug metabolism, systemic administration of 6 delivered 11-fold higher DON exposure to tumor (target tissue; AUC0–t = 5.1 nmol h/g) versus GI tissues (toxicity tissue; AUC0–t = 0.45 nmol h/g). In summary, these studies describe the discovery of a glutamine antagonist prodrug that provides selective tumor exposure.

The Novel Glutamine Antagonist Prodrug JHU395 Has Antitumor Activity in Malignant Peripheral Nerve Sheath Tumor

“…Plates were incubated undisturbed at room temperature for 5 hours. Donor and acceptor samples for prodrugs and positive control were quantified by LC/MS-MS as described below (23)(24)(25).…”

“…We recently developed targeted GA prodrugs around the parent compound DON in attempt to decrease DON exposure to sites of toxicity (e.g., gut) and preferentially deliver DON to target tissues (e.g., nervous tissues, tumors; refs. [23][24][25]. One such targeted GA termed JHU395 (previously 13d) contains (phenyl(pivaloyloxy)methoxy)carbonyl and isopropyl ester modifications to the amine and carboxylate groups of DON, respectively, which significantly increase its lipophilicity relative to DON (clogP 2.75 vs. À2.5; Fig.…”

“…24) as well as GA prodrugs with enhanced DON exposure to lymphoid tumors versus GI tissues in mice (prodrug 6, tumor AUC 0!t ¼ 5.1 nmol/h/g; GI AUC 0!t ¼ 0.45 nmol/ h/g; ratio 11; ref. 25). Thus, prodrug GA may enable improved delivery of active DON to target tissues while avoiding sites of toxicity, enabling better tolerability and translation of this metabolic therapy.…”

The Pleiotropic Effects of Glutamine Metabolism in Cancer

“…Due to the multitude of targets, DON can be efficacious in the treatment of disease, albeit with potentially strong side effects. Efforts to eliminate side effects are ongoing, including development of prodrugs which exhibit minimal release of DON to gastrointestinal tissues or increased release of DON in tumor cells [106,107]. The wide spectrum of enzymes inhibited by DON leads to tempting speculation of disease treatment when the complex metabolic crosstalk of tumor and stroma is taken into consideration.…”

Inhibition of Cancer Metabolism: a Patent Landscape

“…One recently described set of molecules was based around 6-diazo-5-oxo-L-norleucine (DON), one of the oldest known inhibitors of GLS [4]. DON is an irreversible substrate-competitive compound, and is generally toxic, with a number of verified targets, and so even though it has excellent efficacy in cell culture it is unsuitable for use in live subjects [4,58]. However, by preparing a series of prodrugs which would be cleaved to DON in cancer cells, researchers from the Johns Hopkins University demonstrated that compounds such as 19 had similar tissue distribution to DON upon injection in mice, but resulted in much lower plasma concentrations of active DON, suggesting it would have reduced off-target effects [59].…”