Navoximod

Cell experiment:

The IDO inhibitory effect of PEG2k-Fmoc-NLG is tested by an in vitro IDO assay. Briefly, HeLa cells are seeded in a 96-well plate at a cell density of 5000 cells per well and allowed to grow overnight. Recombinant human IFN-γ is then added to each well with a final concentration of 50 ng/mL. At the same time, various concentrations of PEG2k-Fmoc-NLG(L), PEG2k-Fmoc-NLG(S) or Navoximod (NLG919) (50 nM-20 μM) are added to the cells. After 48 h of incubation, 150 μL of the supernatants per well is transferred to a new 96-well plate. Seventy-five μL of 30% trichloroacetic acid is added into each well and the mixture is incubated at 50°C for 30 min to hydrolyse N-formylkynurenine to kynurenine. For colorimetric assay, supernatants are transferred to a new 96-well plate, mixed with equal volume of Ehrlich reagent (2% p-dimethylamino-benzaldehyde w/v in glacial acetic acid), and incubated for 10 min at RT. Reaction product is measured at 490 nm by a plate reader[3].

Animal experiment:

Mice[2]Mice are anesthetized with 4% isoflurane, and the surgical plane of anesthesia is maintained with 2% isoflurane in oxygen. Mice are immobilized in a stereotactic frame for tumor implantation. Briefly, the skull is shaved and exposed with a 0.5 cm skin incision. With antiseptic technique, 105 GL261 cells (suspended in 3 μL RPMI-1640) are injected at the following coordinates with respect to the bregma on the right side (antero-posterior, -2 mm; medio-lateral, 2 mm; dorso-ventral, 3 mm). This placement reproducibly yielded tumor growth in a paracortical area of the posterolateral right frontal lobe. Tumor-bearing mice are treated with combinations of oral DL-1MT (2 mg/mL D-1MT mixed with 2 mg/mL L-1MT) in drinking water, D-1MT (4 mg/mL) in drinking water, Navoximod (6 mg/mL) in drinking water, intraperitoneal cyclophosphamide, intraperitoneal temozolomide, and/or total-body radiation (500 cGy from a 137Cs source), as detailed in figure legends. Mice are observed daily, and sacrificed when they became ill or moribund[2].

References:

[1]. Mario R. Mautino, et al. Abstract 491: NLG919, a novel indoleamine-2,3-dioxygenase (IDO)-pathway inhibitor drug candidate for cancer therapy. AACR 104th Annual Meeting 2013; Apr 6-10, 2013.
[2]. Li M, et al. The indoleamine 2,3-dioxygenase pathway controls complement-dependent enhancement of chemo-radiation therapy against murine glioblastoma. J Immunother Cancer. 2014 Jul 7;2:21.
[3]. Chen Y, et al. An immunostimulatory dual-functional nanocarrier that improves cancer immunochemotherapy. Nat Commun. 2016 Nov 7;7:13443.