Navoximod (GDC-0919) (NLG- 919, GDC-0919) is a potent indoleamine-(2,3)-dioxygenase (IDO) pathway inhibitor (Ki/EC50: 7 nM/75 nM).

IDO:7 nM (Ki), IDO:75 nM (EC50)

在异基因混合淋巴细胞反应(MLR)中，使用表达IDO的人源单核细胞来源的树突状细胞(DCs)，Navoximod强力阻断IDO引起的T细胞抑制并恢复强烈的T细胞反应(ED50：80 nM)。同样，使用来自肿瘤引流淋巴结的表达IDO的小鼠DCs，Navoximod在体外消除IDO引起的特异性T细胞(OT-I)抑制(ED50：120 nM)[1]。Navoximod以浓度依赖的方式抑制IDO活性，其EC50为0.95μM。与游离的Navoximod相比，PEG2k-Fmoc-NLG(L)在抑制IDO方面活性较低(EC50：3.4μM)，而PEG2k-Fmoc-NLG(S)活性最低(EC50>10μM)。将表达IDO的肿瘤细胞与来自BALB/c小鼠的脾细胞共培养会显著抑制T细胞的增殖。这种抑制在用Navoximod处理混合细胞时显著减弱。PEG2k-Fmoc-NLG(L)也能逆转肿瘤细胞的抑制效果，尽管其潜力略低于Navoximod[3]。

在小鼠中，单次口服Navoximod可将血浆和组织中的Kyn浓度降低约50%。体内实验显示，在携带大型B16F10肿瘤的小鼠中，Navoximod的使用显著增强了未激活的pmel-1细胞对含hgp100多肽及CpG-1826的IFA疫苗的抗肿瘤反应。在这个严格的已建立肿瘤模型中，Navoximod加上pmel-1/疫苗的组合在接种后的4天内显著减少肿瘤体积（与仅接受pmel-1/疫苗而不使用Navoximod的对照动物相比，肿瘤体积减少约95%）[1]。当Navoximod与Temozolomide（TMZ）+放射治疗（RT）结合使用时，相对于仅用TMZ+RT治疗的小鼠，Navoximod和1-甲基-D-色氨酸（D-1MT，indoximod）均能提高生存率[2]。

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 the colorimetric assay, supernatants are transferred to a new 96-well plate, mixed with an equal volume of Ehrlich reagent (2% p-dimethylamino-benzaldehyde w/v in glacial acetic acid), and incubated for 10?min at RT. The reaction product is measured at 490?nm by a plate reader [3].

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 the antiseptic technique, 10^5 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; dorsoventral, 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].