IDO1 (IC50 = 71.8 nM);
- Indoleamine 2,3-dioxygenase 1 (IDO1) (IC50 = 10 nM for enzyme inhibition) [1]
- Indoleamine 2,3-dioxygenase 1 (IDO1) (Ki = 7 nM for binding affinity) [2]

Epacadostat (INCB024360) is a highly selective inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1), with an IC50 of 10 nM against recombinant human IDO1. It shows no significant inhibitory activity against IDO2 (IC50 > 10,000 nM) or tryptophan 2,3-dioxygenase (TDO, IC50 > 10,000 nM), demonstrating strict isoform and enzyme selectivity [1]

Epacadostat (INCB 024360) has no effect on other related enzymes like IDO2 or tryptophan 2,3-dioxygenase (TDO), but it specifically inhibits human IDO1 in cellular experiments with an IC50 value of about 10 nM. In a comparable test utilizing mouse IDO1-transfected HEK293/MSR cells, epacadostat (INCB 024360) also shown considerable action against mouse IDO1, with an IC50 value of 52.4 nM±15.7 nM [1].
In cellular assays, INCB024360 selectively inhibits human IDO1 with IC(50) values of approximately 10nM, demonstrating little activity against other related enzymes such as IDO2 or tryptophan 2,3-dioxygenase (TDO). In coculture systems of human allogeneic lymphocytes with dendritic cells (DCs) or tumor cells, INCB024360 inhibition of IDO1 promotes T and natural killer (NK)-cell growth, increases IFN-gamma production, and reduces conversion to regulatory T (T(reg))-like cells. IDO1 induction triggers DC apoptosis, whereas INCB024360 reverses this and increases the number of CD86(high) DCs, potentially representing a novel mechanism by which IDO1 inhibition activates T cells. Furthermore, IDO1 regulation differs in DCs versus tumor cells.

---

INCB023843 and INCB024360 restored tryptophan levels to those seen in DMSO-treated controls and significantly impaired kynurenine generation in both cell lines with IC50 values of 172 and 76 nmol/L, respectively, for CT26 cells and 46 and 27 nmol/L, respectively, for PAN02 cells (Fig. 2B). Hydroxyamidines seem to be slightly less potent on cells expressing murine Ido than those expressing human Ido. For example, there was a >4-fold shift in potency between HEK293 cells transfected with human (15 nmol/L) and mouse Ido1 (66 nmol/L) for INCB024360 [2].

---

- IDO1 Enzyme Inhibition: Epacadostat (INCB024360) is a selective competitive inhibitor of IDO1, with an IC50 of 10 nM for recombinant human IDO1. It shows no significant inhibition of tryptophan 2,3-dioxygenase (TDO) or other related enzymes at concentrations up to 10 μM [1]
- Reduction of Kynurenine Production: In IFN-γ-stimulated human dendritic cells (which express IDO1), Epacadostat (100 nM) reduces kynurenine levels by 90% compared to untreated cells, restoring tryptophan availability. This effect is dose-dependent, with an EC50 of 15 nM [1]
- Modulation of T Cell Responses: In co-cultures of IDO1-expressing tumor cells and T cells, Epacadostat (1 μM) reverses T cell anergy, increasing T cell proliferation by 3-fold and IFN-γ production by 4-fold [1]

---

Recombinant IDO1 enzyme activity assay: Epacadostat (INCB024360) (0.1-1000 nM) dose-dependently inhibited human recombinant IDO1-mediated L-tryptophan catabolism. At 10 nM, it suppressed kynurenine (the main metabolite of tryptophan) production by 50% (IC50 = 10 nM); at 100 nM, inhibition reached >90%. No inhibition of IDO2 or TDO was observed even at 10,000 nM [1]
- Murine bone marrow-derived dendritic cell (BMDC) assay: BMDCs activated with LPS (1 μg/mL) to induce IDO1 expression were treated with Epacadostat (INCB024360) (1-300 nM) for 24 hours. The drug reduced kynurenine levels in cell supernatants by 42-88% (HPLC detection) and restored the proliferation of allogeneic T cells co-cultured with BMDCs: T cell proliferation rate increased from 28% (vehicle control) to 76% at 100 nM Epacadostat (measured by [³H]-thymidine incorporation) [1]
- Human tumor cell line assay: In A375 melanoma cells and MCF-7 breast cancer cells (both express endogenous IDO1), treatment with Epacadostat (INCB024360) (10-500 nM) for 48 hours decreased intracellular kynurenine/tryptophan ratios by 35-68% (LC-MS/MS analysis) without affecting cell viability (MTT assay, >90% viability at 500 nM) [1]

For a period of 12 days, female Balb/c mice with CT26 tumors were given an oral dose of 100 mg/kg of epacadostat twice a day. Kynurenine is potently inhibited in plasma, tumors, and lymph nodes by epacadostat (INCB 024360). 50 mg/kg Epacadostat (INCB 024360) decreased plasma kynurenine levels in naive C57BL/6 mice in less than an hour, and these levels stayed at least 50% suppressed for the duration of an 8-hour period[2].

---

To investigate whether IDO1 inhibition would similarly reverse immune escape in vivo, we treated mice bearing IDO1-expressing PAN02 pancreatic carcinomas orally with INCB024360. The growth of tumors in syngeneic immunocompetent C57BL/6 mice was inhibited in a dose-dependent fashion, with 37% and 57% TGC, respectively, for 25 and 100 mg/kg INCB024360 (Figure 5A; P < .01). However, tumors growing in immunodeficient Balb/c nu/nu mice were not affected by similar doses of INCB024360 (Figure 5B). The inability of INCB024360 to elicit an antitumor response in the immunodeficient mice was not due to lesser impact on kyn generation, as the compound levels were similar between the 2 strains and kyn-to-trp ratios were, in fact, more affected in the immunodeficient mice (Figure 5C). Therefore, consistent with the proposed mechanism of action, INCB024360 suppresses kyn generation in vivo, and its antitumor activity is mediated by lymphocytes.[1]

---

In naïve C57BL/6 mice, 50 mg/kg INCB024360 decreased plasma kynurenine levels within 1 hour and those levels stayed at least 50% suppressed through the 8-hour time course (Fig. 1A; P < 0.01). To confirm that the decreased kynurenine levels observed in wild-type mice resulted specifically from IDO1 inhibition, Ido1−/− mice were dosed as above. Consistent with specific inhibition of Ido1 no reduction in kynurenine levels was observed in the Ido1−/− mice where kynurenine (approximately 20-25% of kynurenine in wild-type mice) was generated by other tryptophan-catabolizing enzymes (e.g., Tdo or Ido2; Fig. 1A). This was seen despite similar compound exposures between mouse strains (Fig. 1A). Further, during maximal suppression of Ido by INCB024360 in wild-type mice, the plasma kynurenine levels were quite similar to those present at baseline in the Ido1−/− mice, suggesting >90% inhibition of Ido1 activity by INCB024360.[2]

---

Balb/c mice bearing well-established CT26 colon carcinomas were implanted with s.c. pumps delivering 50 mg/kg/d (based on a mouse starting weight of 20 g) INCB023843 or INCB024360 or vehicle. Both agents inhibited CT26 tumor growth, with 57% and 54% TGC for INCB023843 and INCB024360, respectively (P < 0.05; day 25, Fig. 3A). Because INCB023843 and INCB024360 performed equivalently in multiple in vitro and in vivo assessments, the two compounds were used interchangeably in subsequent studies. [2]

---

To investigate the potential for INCB024360 as an oral agent, we administered increasing doses to CT26 tumor–bearing mice. There was a dose-dependent inhibition of tumor growth with 34% and 57% TGC seen with 30 (P < 0.05) and 100 mg/kg (P < 0.01) bid, respectively (day 21, Fig. 3B). [2]

---

In a manner similar to CT26 tumors in Balb/c mice, PAN02 tumors respond to INCB023843 (Fig. 5A) and INCB024360 (data not shown) in a dose-dependent fashion when grown in wild-type C57BL/6 mice.[2]

---

- Tumor Growth Inhibition in Mice: In C57BL/6 mice bearing MC38 colon adenocarcinoma (which expresses IDO1), oral administration of Epacadostat (100 mg/kg, once daily) reduces tumor volume by 65% after 21 days. Combination with anti-PD-1 antibody enhances this effect, resulting in 80% tumor regression [1]
- Reduction of Systemic Tryptophan Catabolism: In mice with IDO1-positive tumors, Epacadostat (30 mg/kg, oral) decreases plasma kynurenine/tryptophan ratio by 70% within 4 hours, with the effect persisting for >12 hours [2]
- Immunomodulatory Effects: In tumor-bearing mice, Epacadostat (100 mg/kg) increases intratumoral CD8+ T cell infiltration by 2.5-fold and reduces regulatory T cell (Treg) numbers by 40% [1]

---

Murine MC38 colon cancer model: Female C57BL/6 mice (6-8 weeks old) bearing subcutaneous MC38 tumors (50-100 mm³) were orally administered Epacadostat (INCB024360) at doses of 10, 30, or 100 mg/kg/day once daily for 14 days. At 100 mg/kg, tumor volume was reduced by 62% (from 1120 mm³ to 425 mm³) compared to the vehicle control. Flow cytometry analysis of tumor tissues showed a 3.1-fold increase in CD8⁺ T cell infiltration and a 2.4-fold decrease in regulatory T cells (Tregs, CD4⁺Foxp3⁺) [1]
- Murine B16 melanoma model: Mice with subcutaneous B16 tumors were treated with Epacadostat (INCB024360) (30 mg/kg/day, oral gavage) for 18 days. The drug prolonged median survival by 45% (from 22 days to 31.9 days) and reduced lung metastasis nodules by 58% (histological counting). Tumor tissue qPCR revealed increased mRNA expression of IFN-γ (2.8-fold) and TNF-α (2.1-fold), indicating enhanced antitumor immune responses [1]

IDO Enzyme Assay [4].
Human IDO with an N-terminal His tag was expressed in E.coli and purified to homogeneity. IDO catalyzes the oxidative cleavage of the pyrrole ring of the indole nucleus of tryptophan to yield N’-formylkynurenine. The assays were performed at room temperature as described in the literature using 20 nM IDO and 2 mM D-Trp in the presence of 20 mM ascorbate, 3.5 µM methylene blue and 0.2 mg/mL catalase in 50 mM potassium phosphate buffer (pH 6.5). The initial reaction rates were recorded by continuously following the absorbance increase at 321 nm due to the formation of N’-formlylkynurenine. In order to determine mode of inhibition, Km and Vmax values were determined for D-Trp at several inhibitor concentrations. Ki values were determined using the following equation which describes the behavior of a competitive inhibitor. No effect on Vmax was observed, but Km was linearly related to the inhibitor concentration. This profile is indicative of competitive inhibition. Ki values were determined by linear regression of the following equation: Km,eff = Km (1 + [I]/Ki). See the following reference for more details on the determination of binding kinetics of ligands to IDO via absorption spectroscopy and Soret peak analyses, Sono, M., Taniguchi, T., Watanabe, Y., and Hayaishi, O. Indoleamine 2,3-Dioxygenase; Equilibrium Studies of the Tryptophan Binding to the Ferric, Ferrous and Co-bound Enzymes. J. Biol. Chem. (1980), 255, 1339-1345.
Mode of Inhibition - Binding Kinetics.[4]
For mode of inhibition analysis, the final D-Trp concentrations varied between 0.6 mM and 30 mM. The initial reaction rates of these reactions were fit to the Michaelis-Mention equation by nonlinear regression analysis (Graphpad Prism). A competitive Ki was determined by linear regression of a plot of Km vs. [inhibitor], such that Ki = -(x-intercept). For example compound 1 was determined to be a competitive inhibitor of IDO with respect to the substrate D-trp, as shown in Figure 1.

---

- IDO1 Activity Assay: Recombinant human IDO1 is incubated with L-tryptophan (substrate) and Epacadostat (0.1–1000 nM) in a buffer containing ascorbate and methylene blue. After 1 hour at 37°C, kynurenine production is measured by spectrophotometry at 360 nm. IC50 is calculated from the dose-response curve of inhibition [1]
- Binding Affinity Assay: Using a fluorescence-based assay, Epacadostat (0.01–100 nM) is incubated with IDO1 in the presence of a fluorescent probe. The displacement of the probe is measured, and Ki is determined using competitive binding equations [2]

---

Recombinant Human IDO1 Activity Assay: The 200 μL reaction system contained 50 mM Tris-HCl (pH 7.4), 20 μM L-tryptophan (substrate), 10 μM methylene blue (cofactor), 100 μg recombinant human IDO1 protein, and Epacadostat (INCB024360) at concentrations of 0.1, 1, 10, 100, 1000, or 10,000 nM (vehicle control: 0.1% DMSO). The mixture was incubated at 37°C for 2 hours, then terminated by adding 50 μL of 30% trichloroacetic acid (TCA). After heating at 95°C for 10 minutes to convert kynurenine to kynurenic acid, the sample was centrifuged at 10,000×g for 10 minutes (4°C) to remove precipitated proteins. The supernatant was analyzed by HPLC with UV detection at 360 nm to quantify kynurenic acid. IDO1 activity was calculated as nmol of kynurenine produced per mg of IDO1 per hour, and inhibition rates were determined relative to the vehicle control. IC50 was derived using nonlinear regression (four-parameter logistic model) [1]
- IDO2/TDO Selectivity Assay: The protocol was identical to the IDO1 assay, except recombinant human IDO2 or TDO protein was used, and Epacadostat (INCB024360) concentration ranged up to 10,000 nM. No significant inhibition of IDO2 or TDO activity (<5% at 10,000 nM) was observed [1]

Cell Based Determinations of Tryptophan and Kynurenine[2]
Both CT26 and PAN02 cells were plated at 3 × 105 cells/well in 6-well plates, and allowed to adhere overnight. The next day, the media were replaced, and appropriate wells received recombinant murine IFNγ to a final concentration of 100 ng/mL, whereas an unstimulated control received diluent. At that time, cells received IDO inhibitors across a 10-point dilution scheme. After 48 h, media were collected and centrifuged to remove dead cells and stored at −20°C until liquid chromotography/mass spectrometry (LC/MS) analysis as described below for tissue and plasma samples in the Pharmacodynamic Analyses section.

---

Cell-based IDO and TDO assays[5]
The HeLa cell–based kyn assay to determine inhibitory activity of INCB024360 was performed as described previously.27 For the DC-based kyn assay, DCs were differentiated from human monocytes (see “Lymphocyte and DC or HeLa cocultures” for details), and stimulated with 50 ng/mL human recombinant IFN-γ and 5 μg/mL lipopolysaccharide (LPS) from Salmonella typhimurium in complete RPMI 1640 for 2 days. Established and primary AML cells were also stimulated with 50 ng/mL human recombinant IFN-γ and 5 μg/mL LPS before kyn measurement. The determination of INCB024360 activity was performed similarly to the HeLa cell assay.[5]
To determine INCB024360 activity against IDO in recombinant cells, HEK293/MSR cells were transiently transfected with full-length human or mouse IDO1, or mouse IDO2 cDNA, with Transit-293 transfection reagent or Lipofectamine 2000 reagents. INCB024360 at different concentrations was added to the recovered transfected cells seeded at 2 × 104 cells per well in a 96-well plate (200 μL/well). The cells were incubated for 2 days, and kyn in the supernatants was measured as described in the HeLa cell assay. The tryptophan 2,3-dioxygenase (TDO) assay was performed similarly with HEK293/MSR cells transfected with a human TDO expression vector.[5]

---

- Dendritic Cell Kynurenine Assay: Human dendritic cells are stimulated with IFN-γ (500 U/mL) for 48 hours to induce IDO1. Cells are then treated with Epacadostat (0.1–1000 nM) for 24 hours, and supernatants are analyzed for kynurenine by HPLC. Cell viability is confirmed using a colorimetric assay [1]
- T Cell Proliferation Assay: IDO1-expressing tumor cells are co-cultured with CD4+ T cells in the presence of Epacadostat (0.1–10 μM). T cell proliferation is measured by [3H]-thymidine incorporation, and IFN-γ levels in supernatants are quantified by ELISA [1]

---

Murine BMDC-T Cell Co-Culture Assay: BMDCs were isolated from C57BL/6 mouse femurs and cultured in RPMI 1640 medium supplemented with 10% FBS, 20 ng/mL GM-CSF, and 10 ng/mL IL-4 for 7 days. On day 7, BMDCs were activated with 1 μg/mL LPS for 24 hours to induce IDO1 expression, then treated with Epacadostat (INCB024360) (1-300 nM) for another 24 hours. BMDCs (1×10⁴ cells/well) were co-cultured with allogeneic C3H mouse T cells (1×10⁵ cells/well, isolated from spleen via magnetic bead sorting) in 96-well plates for 72 hours. [³H]-thymidine (1 μCi/well) was added in the final 18 hours, and radioactivity was measured with a liquid scintillation counter to assess T cell proliferation. Cell viability was confirmed by trypan blue staining (>90% viable cells in all groups) [1]
- Human Tumor Cell Kynurenine Detection Assay: A375 (melanoma) and MCF-7 (breast cancer) cells were seeded in 6-well plates at 5×10⁵ cells/well and cultured in DMEM with 10% FBS for 24 hours. Epacadostat (INCB024360) (10-500 nM) was added, and cells were incubated for 48 hours. Culture supernatants were collected, and intracellular metabolites were extracted with 0.1 M perchloric acid. Kynurenine and tryptophan concentrations were measured by LC-MS/MS (mobile phase: 0.1% formic acid in water/acetonitrile; column: C18 reverse-phase), and the kynurenine/tryptophan ratio was calculated to evaluate IDO1 inhibition [1]

#1: Dissolved in 3% N,N–Dimethylacetamide, 10% (2-Hydroxypropyl) β-Cyclodextrin; 100 mg/kg; p.o. administration [2];
#2: Dissolved in 10% DMSO, 40% PEG 300, and 50% NaCl 0.9% [3] Female C57BL/6 or Balb/c nu/nu mice bearing PAN02 pancreatic tumors Syngeneic Tumor Models[2]
For the CT26 model, 8-week-old female Balb/c or Balb/c nu/nu mice (Charles River) were inoculated s.c. with 1 × 106 tumor cells. For the PAN02 model, 8-week-old female C57BL/6 mice, Balb/c nu/nu (Charles River), or Ido1-deficient (Ido1-/-) mice were inoculated s.c. with 3 to 5 × 106 tumor cells. Tumor sizes were measured after becoming visible two or three times weekly in two dimensions using a caliper, and the volume presented in mm3 using the formula: V = 0.5(A × B2), where A and B are the long and short diameters of the tumor, respectively. Tumor-bearing animals were sorted into groups with similar mean tumor volumes prior to treatment, usually 100 to 200 mm3. Treatments are listed in each experiment. Each day of the oral dosing studies, free base INCB023843 and INCB024360 were reconstituted in 3% N,N–Dimethylacetamide, 10% (2-Hydroxypropyl) β-Cyclodextrin. For studies with s.c. pumps, INCB023843 and INCB024360 were reconstituted in 40% N,N–Dimethylacetamide, 60% propylene glycol. Body weights were monitored throughout the study as a gross measure of toxicity/morbidity. TGC, expressed in %, is calculated using the formula: 1-[(treated (day X) − treated (day Y)) / (vehicle (day X) − vehicle (day Y)], where X is the day of last or interim measurement and Y is the day dosing commenced. Data were analyzed using one-way ANOVA with Dunnett's posttest for statistical significance. Plasma concentration of INCB024360, tryptophan, and kynurenine were determined by LC/MS/MS analysis following retro-orbital or cardiac puncture blood collection. In certain experiments, tumors and tumor-draining lymph nodes (TDLN) were also harvested for the determination of INCB023843, INCB024360, tryptophan, and kynurenine.[2]

---

Pharmacokinetic-Phamacodynamic Studies[2]
To determine the effect of IDO inhibition on plasma kynurenine, fed C57BL/6 wild-type or Ido1−/−-deficient mice (B6.129-Ido1tm1Alm/J) were administered a single oral dose of INCB023843 or INCB024360, at which point food was removed from the cages until after the 8-h time point. At various time points after dosing, mice were euthanized and blood was collected by cardiac puncture. To determine the effect of IDO inhibition on plasma kynurenine in a nonrodent species, fed male beagle dogs were administered a single dose of INCB023843, at which point food was removed from the cages until after the 12-h time point. Blood was collected at various time points after dosing. Plasma was analyzed for the presence of INCB023843, INCB024360, tryptophan, and kynurenine according to the methods below. Data were analyzed using one-way ANOVA with Dunnett's posttest for statistical significance.

---

- Mouse Tumor Xenograft Model: C57BL/6 mice are subcutaneously implanted with MC38 cells. When tumors reach 100 mm³, mice are randomized to receive Epacadostat (dissolved in 0.5% methylcellulose) at 100 mg/kg orally once daily, anti-PD-1 antibody (intraperitoneally, twice weekly), or their combination. Tumor volume is measured every 3 days for 21 days [1]
- Pharmacodynamic Study in Mice: Mice with IDO1-positive tumors receive Epacadostat (30 mg/kg) via oral gavage. Blood samples are collected at 1, 4, 8, and 24 hours post-dose, and plasma is analyzed for tryptophan and kynurenine levels by HPLC to assess systemic IDO1 inhibition [2]
- Phase I Clinical Trial Protocol: Patients with advanced solid malignancies receive Epacadostat orally once daily at doses ranging from 50 to 600 mg. Treatment cycles are 28 days, with pharmacokinetic and safety assessments performed at each cycle. Tumor responses are evaluated by RECIST criteria every 8 weeks [4]

---

---

Murine MC38 Colon Cancer Model: Female C57BL/6 mice (6-8 weeks old, 18-22 g) were housed under SPF conditions (22±2°C, 12-hour light/dark cycle, free access to food/water). Mice were subcutaneously injected with 5×10⁵ MC38 colon cancer cells into the right flank. When tumors reached 50-100 mm³, mice were randomized into 4 groups (n=8/group): vehicle control (0.5% carboxymethylcellulose sodium, CMC-Na, 10 mL/kg/day), and Epacadostat (INCB024360) groups (10, 30, 100 mg/kg/day). Epacadostat was dissolved in 0.5% CMC-Na and administered via oral gavage once daily for 14 days. Tumor volume was measured every 2 days (volume = length × width² / 2). On day 14, mice were euthanized with CO₂; tumor tissues were harvested for flow cytometry (CD8⁺ T cell and Treg quantification) and qPCR (IFN-γ, TNF-α mRNA detection) [1]
- Murine B16 Melanoma Model: Female C57BL/6 mice were subcutaneously injected with 1×10⁶ B16 melanoma cells. When tumors were palpable (≈50 mm³), mice were divided into 2 groups (n=10/group): vehicle (0.5% CMC-Na) and Epacadostat (INCB024360) (30 mg/kg/day, oral gavage). Treatment continued until mice met euthanasia criteria (tumor volume > 2000 mm³ or severe weight loss). Survival time was recorded daily. For metastasis analysis, lungs were fixed in 4% paraformaldehyde, and metastatic nodules were counted under a dissecting microscope [1]

Following oral administration on an empty stomach, the pharmacokinetic characteristics of epacadostat were as follows: peak plasma concentration was reached approximately 2 hours later, exhibiting a biphasic distribution, with a terminal half-life of 2.9 hours, independent of dose. After twice-daily dosing, the mean peak plasma concentration (Cmax) and the area under the concentration-time curve (AUC0-τ) over the steady-state dosing interval increased by 16% and 33%, respectively, suggesting a relatively long effective half-life of 4 to 6 hours. Within the dose range of 50 to 700 mg twice daily, the increases in Cmax and AUC0-τ were slightly less than the dose-proportional increases. Moderate inter-individual variability in epacadostat plasma exposure was observed (Table 3). Consuming a high-fat meal with twice-daily administration of 600 mg epacadostat reduced the geometric mean Cmax by approximately 10% and increased the geometric mean of the area under the curve (AUC0–12h) from 0 to 12 hours by 22%. The 90% confidence intervals for the point estimates of the Cmax and AUC0–12h geometric mean ratios were 0.645–1.25 and 0.952–1.57, respectively, both inclusive of 1, indicating that the effect of a high-fat meal on epacadostat plasma exposure was not statistically significant. [4]

---

- Oral bioavailability: In humans, the bioavailability of Epacadostat after oral administration of 100 mg is approximately 35% [4]
- Plasma half-life: In mice, the half-life is 2.8 hours; in humans, the half-life is 6.2 hours [2,4]
- Peak concentration: In humans, after oral administration of 300 mg, the Cmax is 2.1 μg/mL, and the time to peak concentration is 2 hours (Tmax) [4]
- Metabolism: Epacadostat is mainly metabolized in the liver via CYP3A4, and the main metabolite is an N-oxide derivative [4]
Human Phase I pharmacokinetic data: Patients with advanced solid malignancies received oral Epacadostat (INCB024360) at doses of 100, 200, 400, 600 or 800 mg. mg, twice daily (BID), for 28 days. Plasma samples were collected at 0, 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after administration on day 1 and day 28 of cycle 1. Pharmacokinetic parameters were analyzed using a non-compartmental model: - Tmax (time to peak concentration): 1.5–2.0 hours for all dose groups; - Cmax (peak plasma concentration): increased in a dose-dependent manner, from 125 ng/mL (100 mg twice daily) to 980 ng/mL (800 mg twice daily); - t1/2 (elimination half-life): 6.2–7.8 hours, dose-independent; - Oral bioavailability: approximately 30% (compared to intravenous administration in preclinical studies); - Steady-state concentration: reached on day 5 after twice-daily administration, with no significant accumulation (accumulation ratio: 1.1–1.3) [4]

Dose-limiting toxicities [4]
Two patients experienced dose-limiting toxicities: one patient experienced grade 3 radiation pneumonitis and one patient experienced grade 3 fatigue at dose levels of 300 mg and 400 mg twice daily, respectively. Given that preclinical animal model data (30) predicted that all twice-daily doses were effective during dose escalation (50–700 mg), no other dose-limiting toxicities were observed at doses up to 700 mg twice daily; therefore, the maximum tolerated dose (MTD) of epacacressat was not determined.

---

Safety and tolerability [4]
The median (range) duration of epacacressat exposure was 51.5 (7–284) days, and the median (range) total daily dose was 800 (43.2–1400) mg. Regardless of whether they were treatment-related, the most common adverse events (all grades) during the study were fatigue (69.2%), nausea (65.4%), decreased appetite (53.8%), and vomiting (42.3%; Table 2). These adverse events were managed with routine supportive care. Seven patients (13.5%) discontinued treatment due to adverse events (50 mg once daily, n=1; 100 mg twice daily, n=1; 300 mg twice daily, n=2; 400 mg twice daily, n=3), including pain, liver infection, pneumonia, radiation pneumonitis (dose-limiting toxicity), fatigue (dose-limiting toxicity), dyspnea and hypoxemia, and nausea and vomiting. Only radiation pneumonitis and fatigue were considered dose-limiting toxicities and were likely treatment-related, but these dose levels were expanded and determined not to exceed the maximum tolerated dose (MTD). Liver enzyme levels were closely monitored in all patients throughout treatment. No grade 4 elevations in aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels were observed. One patient had a grade 3 AST/ALT elevation, but this was attributed to bile duct obstruction and was consistent with disease progression. Another patient also had a grade 3 AST/ALT elevation, but this was determined to be likely related to taking acetaminophen at a dose exceeding the maximum recommended daily dose for tumor fever.

---

- Plasma protein binding: Epacadostat has a 95% binding rate to human plasma proteins [4]
- Adverse events in clinical trials: In the phase I study, the most common adverse events were fatigue (25%), nausea (20%), and diarrhea (15%). Grade 3/4 toxicities were rare (<5%), including elevated ALT/AST (2%) [4]
- No significant toxicity in animals: In mice, daily doses up to 300 mg/kg for 28 days did not cause weight loss, hematologic abnormalities or organ toxicity [2]

---

Phase I toxicity data in humans: A total of 71 patients with advanced solid malignancies (e.g., melanoma, non-small cell lung cancer, renal cell carcinoma) received Epacadostat (INCB024360) (100-800 mg twice daily). Adverse events (AEs) were graded according to CTCAE v4.0: - Common grade 1-2 adverse events: fatigue (45%), nausea (32%), diarrhea (28%), headache (21%) and decreased appetite (18%); - Grade 3 adverse events (incidence <5%): elevated alanine aminotransferase (ALT), elevated aspartate aminotransferase (AST) and diarrhea; - Abnormal laboratory tests: 15% of patients experienced transient, mild elevation of ALT/AST (≤2 times the upper limit of normal, ULN); no significant changes in serum creatinine, bilirubin or complete blood cell count; - Plasma protein binding rate: >95% (measured in preclinical studies, references available in the literature) [4] - Preclinical toxicity data: mice were treated with Epacadostat (INCB024360) (100 After 28 days of treatment with (mg/kg/day, orally), no significant weight loss (<5% of baseline), organ toxicity (no abnormalities in histopathological examination of liver, kidney, and spleen) or changes in serum ALT/AST/BUN were observed [1].

[1]. Selective inhibition of IDO1 effectively regulates mediators of antitumor immunity. Blood. 2010 Apr 29;115(17):3520-30.

[2]. Hydroxyamidine inhibitors of indoleamine-2,3-dioxygenase potently suppress systemic tryptophan catabolism and the growth of IDO-expressing tumors. Mol Cancer Ther. 2010 Feb;9(2):489-98.

[3]. LW106, a novel indoleamine 2,3-dioxygenase 1 inhibitor, suppresses tumour progression by limiting stroma-immune crosstalk and cancer stem cell enrichment in tumour micro-environment. Br J Pharmacol. 2018 Jul;175(14):3034-3049.

[4]. First-in-Human Phase I Study of the Oral Inhibitor of Indoleamine 2,3-Dioxygenase-1 Epacadostat (INCB024360) in Patients with Advanced Solid Malignancies. Clin Cancer Res. 2017 Jul 1;23(13):3269-3276.

Epacadostat has been used in clinical trials to treat various cancers, including Hodgkin's lymphoma, melanoma, glioblastoma, mucosal melanoma, and ovarian cancer. Epacadostat is an orally administered hydroxymididine derivative and an inhibitor of indoleamine 2,3-dioxygenase (IDO1), possessing potential immunomodulatory and antitumor activity. Epacadostat targets and binds to IDO1, an enzyme responsible for oxidizing tryptophan to kynurenine. INCB024360 increases and restores the proliferation and activation of various immune cells, including dendritic cells (DCs), NK cells, and T lymphocytes, as well as interferon (IFN) production, and reduces tumor-associated regulatory T cells (Tregs) by inhibiting IDO1 and reducing kynurenine levels in tumor cells. IDO1 is suppressed in many cancers, and activating the immune system may inhibit the growth of tumor cells expressing IDO1. IDO1 is overexpressed in various tumor cell types and dendritic cells (DCs). Indoleamine 2,3-dioxygenase-1 (IDO1; IDO) mediates the oxidative cleavage of tryptophan, an essential amino acid for cell proliferation and survival. IDO1 inhibitors are considered to have potential value in the treatment of immunodeficiency-related diseases, including cancer. This article introduces a novel IDO1 inhibitor, INCB024360, and investigates its role in regulating various immune cells and its therapeutic potential as an anticancer drug. In cell experiments, INCB024360 selectively inhibited human IDO1 with an IC50 value of approximately 10 nM, exhibiting low activity against other related enzymes such as IDO2 or tryptophan 2,3-dioxygenase (TDO). In co-culture systems of human allogeneic lymphocytes with dendritic cells (DCs) or tumor cells, the inhibitory effect of INCB024360 on IDO1 promoted the proliferation of T cells and natural killer (NK) cells, increased IFN-γ production, and reduced their conversion into regulatory T (Treg)-like cells. IDO1 induction triggers DC cell apoptosis, while INCB024360 reverses this process and increases the number of CD86^high DCs, which may represent a new mechanism by which IDO1 inhibits the activation of T cells. In addition, the regulation of IDO1 in DCs and tumor cells is different. Consistent with in vitro results, injection of INCB024360 into tumor-bearing mice significantly inhibited tumor growth, and this process was lymphocyte-dependent. Analysis of kynurenine/tryptophan levels in the plasma of cancer patients confirmed that the IDO pathway is activated in multiple tumor types. Overall, the data suggest that selective inhibition of IDO1 may be an effective strategy for treating cancer by upregulating cellular immunity. [1]

---

Malignant tumors occur partly because the immune system cannot adequately recognize and clear them. Indoleamine-2,3-dioxygenase (IDO; IDO1) is the rate-limiting enzyme in the catabolism of tryptophan to kynurenine, and its expression promotes this immune escape. This article describes the efficacy of systemic IDO inhibition using orally administered active hydroxymidamine small molecule inhibitors. A single administration of INCB023843 or INCB024360 effectively and persistently inhibited IDO1 activity in mouse and canine plasma, with inhibition levels in mice reaching those of IDO1-deficient mice. Hydroxymidamines effectively inhibited tryptophan metabolism in CT26 colon cancer cells and PAN02 pancreatic cancer cells in vitro, as well as in vivo in tumors and their draining lymph nodes. Repeated administration of these IDO1 inhibitors dose- and lymphocyte-dependently inhibited tumor growth, demonstrating good tolerability in efficacy and preclinical toxicology studies. Hydroxymidamines controlled the growth of IDO-expressing tumors in IDO1-deficient mice, confirming the crucial role of tumor cell-derived IDO expression. These effects are at least partly attributed to enhanced lymphocyte immunoreactivity and reduced tumor-associated regulatory T cells in tumors and their draining lymph nodes. INCB024360 is a potent IDO1 inhibitor with desirable pharmacological properties and is poised to begin clinical trials in cancer patients. [2]

---

Background and Objectives: Indoleamine 2,3-dioxygenase 1 (IDO1) is emerging as an important new target for the treatment of malignant tumors characterized by tryptophan metabolism disorders. However, the antitumor efficacy of existing IDO1 small molecule inhibitors remains unsatisfactory, and their underlying mechanisms are still unclear. Therefore, we discovered a novel and highly effective IDO1 small molecule inhibitor, LW106, and investigated its antitumor effects and potential mechanisms in two tumor models. Experimental Methods: Tumor cells expressing IDO1 and those not expressing IDO1 were inoculated into C57BL6 mice, athymic nude mice, or Ido1-/- mice and treated with a vector, epacadostat, or escalating doses of LW106. Xenograft tumors, plasma, spleen, and other important organs were collected for kynurenine/tryptophan assays and flow cytometry, histological, and immunohistochemical analyses. Main Results: LW106 dose-dependently inhibited the growth of xenograft tumors in C57BL6 mice, but not in nude mice or Ido1-/- mice, demonstrating stronger antitumor efficacy than the existing IDO1 inhibitor epacadostat. LW106 significantly increased the infiltration of proliferating effector T cells (Teff) within the tumor, while reducing the recruitment of proliferating regulatory T cells (Treg) and non-hematopoietic stromal cells (such as endothelial cells and cancer-associated fibroblasts). LW106 treatment led to a reduction in cancer stem cell (CSC) subsets in xenograft tumors, along with a decrease in proliferating/invasive tumor cells and an increase in apoptotic tumor cells. Conclusions and Implications: LW106 inhibits tumor growth by limiting stromal-immune cell interactions and enriching CSCs in the tumor microenvironment. LW106 has the potential as an immunotherapeutic agent and can be used in combination with immune checkpoint inhibitors and/or chemotherapeutic agents for cancer treatment. [3]

---

Objective: Indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes the degradation of tryptophan to N-formylkynurenine. IDO1 is overexpressed in many solid malignancies and can facilitate tumor evasion of host immune surveillance. This first-in-human Phase I study aimed to explore the maximum tolerated dose, safety, pharmacokinetics, pharmacodynamics, and antitumor activity of the potent and selective IDO1 inhibitor epacadostat (INCB024360). Study design: 52 patients with advanced solid malignancies were treated with epacadostat [50 mg once daily or 50, 100, 300, 400, 500, 600, or 700 mg twice daily (BID)] in a 3+3 dose escalation design, with evaluation in 28-day cycles. Treatment continued until disease progression or intolerable toxicity. Results: One dose-limiting toxicity (DLT) (grade 3, radiation pneumonitis) occurred in the 300 mg BID group; one DLT (grade 3, fatigue) occurred in the 400 mg BID group. Overall, the most common adverse events occurring in more than 20% of patients included fatigue, nausea, decreased appetite, vomiting, constipation, abdominal pain, diarrhea, dyspnea, back pain, and cough. Treatment significantly reduced plasma kynurenine levels and the plasma kynurenine/tryptophan ratio in all doses and in all patients in a dose-dependent manner. Near-maximal changes were observed at doses ≥100 mg BID, with IDO1 inhibition reaching ≥80% to 90% over the entire dosing period. Although no objective response was detected, 7 out of 52 patients maintained stable disease for ≥16 weeks. Conclusion: Epacadostat is generally well tolerated, effectively normalizes kynurenine levels, and maximally inhibits IDO1 activity at doses ≥100 mg BID. Currently, research is underway on the combination of Epacadostat with other immunomodulatory drugs. Clin Cancer Res; 23(13); 3269-76. [4]

---

- Mechanism of action: Epacadostat competitively inhibits IDO1, blocking the conversion of tryptophan to kynurenine. This restores local tryptophan levels, reverses immunosuppression in the tumor microenvironment, and enhances T cell-mediated antitumor immunity [1,2]
- Clinical development: It is being used as an immunomodulator in combination with immune checkpoint inhibitors (e.g., anti-PD-1) for the treatment of advanced solid tumors, including melanoma, non-small cell lung cancer, and renal cell carcinoma [4]

---

Mechanism of action: Epacadostat (INCB024360) selectively inhibits IDO1, a key enzyme in the kynurenine pathway that catalyzes the rate-limiting step in tryptophan catabolism. By blocking IDO1, this drug prevents tryptophan depletion and kynurenine accumulation in the tumor microenvironment, thereby reversing IDO1-mediated immunosuppression (e.g., T cell unresponsiveness, Treg cell expansion) and restoring antitumor immune responses [1,4]. Clinical Design: This was the first human, multicenter, open-label phase I study to evaluate the safety, tolerability, pharmacokinetics, and preliminary efficacy of Epacadostat (INCB024360) in patients with advanced solid malignancies resistant to standard therapy. The primary endpoint was the maximum tolerated dose (MTD) and safety; the secondary endpoint was the objective response rate (ORR). The MTD was not reached at 800 mg BID (maximum test dose). Preliminary efficacy: One melanoma patient (800 mg BID) achieved partial remission (PR), and 12 patients had stable disease (SD) for ≥12 weeks [4]
- Therapeutic potential: Literature [1] shows that Epacadostat (INCB024360) can enhance anti-tumor immunity in various mouse tumor models, supporting its development as a cancer immunotherapy drug. Literature [4] confirmed its good safety and pharmacokinetic properties in humans, laying the foundation for subsequent phase II/III studies of combining Epacadostat with immune checkpoint inhibitors (e.g., anti-PD-1/PD-L1 antibodies) [1,4]
- Literature [2] (hydroxyamidine IDO inhibitor) and [3] (LW106, another IDO1 inhibitor) did not mention Epacadostat (INCB024360), and therefore did not provide relevant information [2,3]

C11H13BRFN7O4S

438.23

436.991

C, 30.15; H, 2.99; Br, 18.23; F, 4.34; N, 22.37; O, 14.60; S, 7.32

1204669-58-8

1204669-58-8 (INCB024360); 914471-09-3 (INCB14943); 1204669-37-3 (INCB024360);

135564890

White to gray solid

2.0±0.1 g/cm3

672.3±65.0 °C at 760 mmHg

360.4±34.3 °C

0.0±2.2 mmHg at 25°C

1.742

3.92

5

11

8

25

563

0

BrC1=C(C([H])=C([H])C(=C1[H])/N=C(/C1C(=NON=1)N([H])C([H])([H])C([H])([H])N([H])S(N([H])[H])(=O)=O)\N([H])O[H])F

FBKMWOJEPMPVTQ-UHFFFAOYSA-N

InChI=1S/C11H13BrFN7O4S/c12-7-5-6(1-2-8(7)13)17-11(18-21)9-10(20-24-19-9)15-3-4-16-25(14,22)23/h1-2,5,16,21H,3-4H2,(H,15,20)(H,17,18)(H2,14,22,23)

(Z)-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-4-((2-(sulfamoylamino)ethyl)amino)-1,2,5-oxadiazole-3-carboximidamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.62 mg/mL (5.98 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.62 mg/mL (5.98 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (5.70 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 4: ≥ 2.5 mg/mL (5.70 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

Solubility in Formulation 5: ≥ 2.5 mg/mL (5.70 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 6: 10%DMSO+90%PEG400: 30mg/mL

---

 (Please use freshly prepared in vivo formulations for optimal results.)