Novel lymphoid targeted prodrugs of the glutamine antagonist DON for the treatment of hematological malignancies

Objective The glutamine antagonist 6‐diazo‐5‐oxo‐l‐norleucine (DON) has shown robust efficacy in both lymphoma and leukemia animal models and exploratory clinical studies. DON broadly blocks glutamine utilizing reactions critical for the synthesis of nucleic acids/proteins and the generation of alpha‐ketoglutarate for energy metabolism. Unfortunately its severe toxicity hampered its development. Using a prodrug approach, we aimed to synthesize lymphoid‐targeted prodrugs of DON designed to circulate intact in blood and preferentially activate inside PBMCs affording reduced systemic exposure and improved tolerability, while maintaining its robust efficacy. methods Synthetic efforts for DON prodrugs were particularly challenging due to DON's inherent instability. We found it was imperative to mask both the carboxylate as well as the amine functionality of DON to achieve sufficient chemical stability. We concealed the carboxylate and amine with various alkyl esters (e.g. methyl, ethyl and isopropyl), amino acids (e.g. leucine, alanine), and other bulkier promoeities used in FDA approved drugs (e.g. POC, POM, ODOL). We then evaluated the prodrugs for in vitro plasma stability across multiple species (mouse, dog, monkey, pig, human). Ex vivo whole blood experiments were further conducted to determine selective conversion in PBMCs. Lastly in vivo experiments using pigs were carried out and PBMC to plasma ratios were calculated. results Our novel DON prodrugs showed species specific plasma stability with rodents metabolizing the prodrug more rapidly compared to higher species. Of the various modifications on the carboxylate, the isopropyl ester afforded significant lymphoid‐targeting in pigs, monkeys, dog, and humans. In human whole blood ex vivo studies several of the isopropyl ester containing prodrugs delivered DON into PBMCs versus plasma with a >30‐fold differential, in contrast to DON which delivered a small percentage (<10%) of drug to PBMCs. Lastly in vivo studies in pigs confirm this PBMC selectivity with favorable pharmacokinetics. conclusions We have successfully synthesized lymphoid‐targeted DON prodrugs which deliver >30‐fold more DON to lymphoid cells in human whole blood versus plasma. Comparative efficacy and toxicity studies are ongoing in lymphoma animal models. These novel prodrugs may pave a path for the development of a new and safer therapeutic for hematological malignancies.