dental JHU395 delivered DON to tumor tissues

dental JHU395 delivered DON to tumor tissues. of the RAS-GAP and presents with a wide range of manifestations including multiple cancers (12). MPNST is definitely poorly responsive GSK2593074A to chemotherapy and radiation therapy, and the only chance for long term survival is definitely if the lesion is completely resected with wide margins (13, 14). As MPNST most often evolves in essential, deep areas such as major nerve plexi, this is hardly ever feasible and hence there is an outsized chance of mortality from MPNST versus additional sarcomas (15). Small molecule glutamine antagonists (GA) are important tools for studying glutamine metabolism and have garnered interest as antitumor therapeutics. 6-diazo-5-oxo-L-norleucine (DON) and its naturally happening prodrug azotomycin are diazo analogs of glutamine that irreversibly inhibit multiple glutamine-utilizing enzymes. GA have been observed to cause decreased proliferation or induction of apoptosis, depending on genetic context, cell/cells type, and environment (16, 17, 18). GA have been tested clinically (19), including for sarcomas (20). Regrettably, their clinical development was hampered by use of high intermittent GSK2593074A dosing regimens poorly suited to a metabolic inhibitor (19, 21) and gastrointestinal (GI) toxicity, as the GI system is highly glutamine utilizing (22). We recently developed targeted GA prodrugs round the parent compound DON in attempt to decrease DON exposure to sites of toxicity (e.g. gut) and preferentially deliver DON to target cells (e.g. nervous cells, tumors) (23C25). One such targeted GA termed JHU395 (previously 13d) consists of (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 ?2.5) (Figure 1C) (23). Prior investigation of JHU395 shown the prodrug remains intact in swine and human being plasma (83C88% at 1 hour) (23). When directly compared to an equimolar dose of DON given to swine, JHU395 resulted in significantly lower DON exposure in plasma (AUC0t 29.9 nmol?hr/ml vs 5.7 nmol?hr/ml), leading to a nearly 10-fold enhancement of the DON brain-to-plasma percentage (DON brain-to-plasma 0.148; JHU395-derived DON brain-to-plasma 1.38; percentage 9.3) (23). Our group has also identified additional GA prodrugs with enhanced DON CSF-to-plasma percentage in non-human primates (compound 5c, DON CSF-to-plasma percentage 0.038; 5c-derived DON CSF-to-plasma percentage 0.28; percentage 7.6) (24) as well while GA prodrugs with enhanced DON exposure to lymphoid tumors versus GI cells in mice (prodrug 6, tumor AUC0t=5.1 nmol?h/g; GI AUC0t=0.45 nmol?h/g; percentage 11) (25). Therefore prodrug GA GSK2593074A may enable improved delivery of active DON to target cells while avoiding sites of toxicity, enabling better tolerability and translation of this metabolic therapy. Open in a separate window Number 1: MPNST cells are sensitive to glutamine deprivation and competitive glutamine antagonists. A) Live cell counts from trypan blue exclusion assay comparing growth of human being MPNST cells (sNF96.2) in control ( 2mM glutamine) or low (50M) glutamine press normalized to quantity of cells seeded. Cells were counted every 24 hours for five days. Data is definitely +/? S.D. B) Percent growth of cells based on alamar blue fluorescence (590 nm) of human being MPNST or immortalized Schwann cells (ipn02.32; imm SC) growing in glutamine-free press with added glutamine titrated at increasing concentrations. Viability was measured at 72 hours glutamine treatment. Data is definitely +/? S.D. C) Constructions of DON and JHU395 with clogP ideals indicated. D) Percent viable MPNST and immortalized Schwann cells based on alamar GSK2593074A blue fluorescence normalized to untreated controls following treatment with DON or JHU395. Viability was measured at 72 hours drug treatment. Data is definitely +/? S.D. *** 0.0001 p 0.001, ** 0.001 p 0.01, * GSK2593074A 0.01 p 0.05 by Students t-test (A). There has been desire for metabolic inhibitors as therapies for MPNST (26), but glutamine rate of metabolism has only been explored using the selective glutaminase inhibitor CB-839 (27). We hypothesized that a broadly active GA with target cells penetration could provide further insight into MPNST glutamine rate of metabolism. Compared to immortalized healthy Schwann cells, we found that human being NF1-connected MPNST cells were sensitive to glutamine GNGT1 deprivation and antagonism. JHU395 was stable in human being plasma and delivered DON to human being MPNST cells in tradition and to a murine flank MPNST. We then recognized a daily oral dosing routine of JHU395 that was well tolerated and delivered micromolar levels of DON.