Aryl hydrocarbon receptor (AHR) - binds to AHR and displaces ligands. IC50 (AHR radioligand binding competition assay) = 0.5 nM. [2]
IC50 (HepG2 DRE-luciferase reporter assay with VAF347 agonist) = 91 nM. IC50 with kynurenine = 89 nM; with kynurenic acid = 30 nM; with ITE = 68 nM. Basal activity IC50 = 14 nM. [2]
IC50 (Hepa1.6 mouse cell line Cyp1a1 luciferase assay with VAF347) = 36 nM. [2]
IC50 (H411E rat cell line Cyp1a1 activity assay with kynurenine) = 151 nM. [2]
IC50 (cynomolgus monkey PBMC CYP1B1 qRT-PCR) = 6.2 nM. [2]

- In human HepG2 cells expressing an AHR-dependent DRE-luciferase reporter, IK-175 inhibited luciferase expression stimulated by the AHR agonist VAF347 (80 nM) with an IC50 of 91 nM. Similar inhibition was observed with other AHR agonists: kynurenine (200 μM, IC50 = 89 nM), kynurenic acid (200 μM, IC50 = 30 nM), and ITE (20 nM, IC50 = 68 nM). IK-175 also inhibited basal AHR activity with an IC50 of 14 nM, with no agonist activity. [2]
- In a photoaffinity ligand competition assay using humanized AHR mouse liver cytosol, IK-175 displaced a radiolabeled dioxin analog from AHR with an IC50 of 0.5 nM, compared to 9.0 nM for the tool inhibitor CH-223191. [2]
- In mouse Hepa1.6 cells, IK-175 inhibited VAF347 (2 μmol/L)-stimulated Cyp1a1-mediated luciferase activity with an IC50 of 36 nM. [2]
- In rat H411E cells, IK-175 inhibited kynurenine (100 μmol/L)-stimulated Cyp1a1 activity with an IC50 of 151 nM. [2]
- In cynomolgus monkey PBMCs, IK-175 inhibited AHR-dependent CYP1B1 gene expression with an IC50 of 6.2 nM. [2]
- In selectivity screening, IK-175 (1 μmol/L) showed limited activity against a panel of 87 receptors, transporters, and enzymes. In a screen of 371 kinases at 10 μmol/L, only 6 kinases showed >65% inhibition; IC50 values for these kinases (CDK2/cyclin E, CDK2/cyclin E2, CHK2, CK1d, DAPK1, SRPK2) ranged from approximately 2 to 9 μmol/L. IK-175 showed no agonistic activity against PXR and only minimal inhibitory activity at concentrations above 3 μmol/L. [2]
- IK-175 had no direct growth inhibitory effects on 100 human cancer cell lines or on murine cancer cell lines CT26 and B16-IDO1. [2]
- In activated human CD3+/CD28+ T cells, IK-175 inhibited CYP1A1 and IL22 gene expression with IC50 values of 11 and 30 nM, respectively, and inhibited IL22 cytokine production with an IC50 of 7 nM while increasing IL2 production approximately 2-fold. [2]
- In a human Th17 differentiation assay, IK-175 (3 μM) led to an increase in IL17A+IL22- expressing cells and a decrease in IL17A+IL22+ expressing cells compared to Th17 alone. The AHR agonist ITE (3 μM) showed the opposite effect. [2]

The statistical significance of tumor growth inhibition was shown when AHR antagonist 5 was utilized alone. In comparison to anti-PD-1 alone, the combination of AHR antagonist 5 (10 mg/kg; oral; given daily for 3 weeks) with anti-PD-1 markedly suppressed tumor development [1].

---

- Pharmacodynamic study in mice: In female Balb/c mice, oral administration of IK-175 (5, 10, or 25 mg/kg) along with the AHR agonist VAG539 (30 mg/kg, prodrug of VAF347) dose-dependently inhibited AHR-dependent Cyp1a1 gene expression in liver and spleen at 4 and 10 hours. At 4 hours, inhibition in the liver was 78%, 93%, and 98% for 5, 10, and 25 mg/kg, respectively. In the spleen, inhibition was 35%, 76%, and 97%, respectively. The 25 mg/kg dose maintained >94% inhibition at 10 hours. [2]
- B16-IDO1 syngeneic melanoma model: C57BL/6 mice bearing B16-IDO1 tumors were treated with IK-175 (25 mg/kg, oral, once daily). Tumor growth inhibition with IK-175 alone was modest but not statistically significant. Combination with anti-PD-1 antibody (10 mg/kg, i.p., every 3 days for 5 doses) resulted in significantly increased antitumor effect compared to either single agent alone, including one complete response. No body weight loss was observed. [2]
- CT26 syngeneic colon carcinoma model: Balb/c mice bearing CT26 tumors (which endogenously express high IDO1) were treated with IK-175 (25 mg/kg, oral, once daily). IK-175 as a single agent significantly inhibited tumor growth (P = 0.0015). Combination with anti-PD-1 (10 mg/kg, i.p., twice weekly for 5 doses) resulted in 7 complete responses and enhanced survival. Mice with complete responses rejected tumor rechallenge, indicating immune memory. No body weight loss was observed. [2]
- Combination with liposomal doxorubicin: In CT26 tumor-bearing mice, liposomal doxorubicin treatment increased AHR pathway activation (Cyp1b1 and Ido1 expression). Combination of IK-175 (25 mg/kg, oral, once daily) with liposomal doxorubicin (1 mg/kg, i.v., once weekly) led to significant tumor growth inhibition compared to liposomal doxorubicin alone, with one complete response and enhanced survival. Similar combination activity was observed in the MC38 syngeneic colon cancer model. [2]
- Tumor immune microenvironment changes: In CT26 tumor-bearing mice treated with IK-175 (25 mg/kg, oral, once daily for 7 days), analysis of tumor-draining lymph nodes showed higher levels of CD8+ T cells producing IL-2, TNFα, and IFNγ. The CD8+:Treg ratio increased significantly. Analysis of the tumor immune infiltrate showed an increase in proinflammatory M1 macrophages (MHCII+ CD206-), shifting the overall M1:M2 ratio. [2]
- Phase I clinical trial: In a first-in-human study, IK-175 was administered orally at doses ranging from 200 mg to 1,600 mg daily (doses above 1,200 mg were given every 12 hours) as a single agent and in combination with nivolumab (480 mg i.v. every 4 weeks) in patients with advanced solid tumors and urothelial carcinoma. The recommended phase 2 dose was established. Target engagement was confirmed by ex vivo AHR activation assays and dose-dependent modulation of CYP1B1 expression in blood (>85% reduction at 1,200 mg). In the single-agent arm, 1/13 patients with urothelial carcinoma achieved a confirmed partial response (ORR 7.7%) lasting 22.6 months. In the combination arm, 2/33 patients achieved confirmed partial responses (ORR 6.1%) with response durations of 4.4 and 7.3 months. Clinical benefit (stable disease for >16 weeks) was observed in multiple patients. [3]

- HepG2 DRE-luciferase reporter assay: HepG2 cells transiently transfected with the DRE-Luc reporter were plated. Dilutions of IK-175 or DMSO control and 80 nmol/L VAF347 (AHR agonist) were added in triplicate and incubated for 6 hours. Media was removed, Bright Glo reagent added, and luminescence read on a plate reader. IC50 values were calculated. [2]
- AHR radioligand binding competition assay: Cytosol from humanized AHR mouse liver was prepared. A photoaffinity ligand (2-azido, 3 iodo 125I, 7,8-dibromo-dibenzo-p-dioxin) was used. IK-175 and CH223191 solutions were added to liver cytosol plus photoaffinity ligand, incubated 30 minutes at room temperature, placed on ice, then charcoal/dextran added. Samples were centrifuged, exposed to UV light, run on SDS-PAGE, and visualized by autoradiography. Radioactive bands were excised and counted in a gamma counter. IC50 values were determined. [2]
- Murine Hepa1.6 and rat H411E Cyp1a1 luciferase reporter assay: H411E or Hepa1.6 cells were plated and cultured overnight. Dilutions of IK-175 and agonist (100 μmol/L kynurenine for H411E, 2 μmol/L VAF347 for Hepa1.6) were added and incubated 24 hours. Cyp1a1 enzyme activity was measured using the p450-Glo Assay; luminescence was quantified. [2]
- Cynomolgus monkey PBMC CYP1B1 Quantigene Plex assay: Cryopreserved cynomolgus macaque PBMCs were cultured in RPMI medium with 10% FBS. Cells were treated with IK-175 (4.5 nmol/L to 10 μmol/L) in duplicate with 0.1% DMSO for 24 hours at 37°C. Cells were lysed and stored until Quantigene Plex assay analysis using a custom panel with probes for primate CYP1B1, AHRR, and housekeeping genes PPIB and B2M. Mean fluorescence activity was quantified on a Luminex FlexMap3D instrument. [2]

- HepG2 DRE-luciferase reporter assay: HepG2 cells were transiently transfected with the DRE-Luc reporter. Cells were treated with IK-175 dilutions and 80 nmol/L VAF347 for 6 hours. Luminescence was read after adding Bright Glo reagent. [2]
- AHR binding assay: The assay used cytosol from humanized AHR mouse liver. IK-175 was tested for its ability to displace a radiolabeled photoaffinity ligand from AHR. Samples were run on SDS-PAGE, visualized by autoradiography, and radioactive bands were counted. [2]
- Human Pan-T-cell activation assay: Cryopreserved pan-T cells were activated with human CD3/CD28 T-cell activator and cultured in T-cell medium at 50,000 cells/well. Cells were treated with DMSO and different concentrations of IK-175 in duplicate. After 24 hours, cDNA was collected using Cells-to-CT Kit. RT-PCR was conducted using Taqman fast advanced master mix with human CYP1A1, IL22, and B2M primers. After 48 hours, supernatant was analyzed for IL22 and IL2 using MSD V-plex plates. [2]
- Human Th17 differentiation assay: Naive human CD4+/CD62L+ T cells were isolated from cryopreserved CD4+ T cells by magnetic selection. Cells were activated with CD3/CD28 T-cell activator and differentiated with human Th17 cytokines (50 ng/mL IL6, 20 ng/mL IL1β, 10 ng/mL IL23, 1 ng/mL TGFβ, 12 μg/mL anti-IFNγ antibody, 10 μg/mL anti-IL4 antibody) for 11 days. Cells were treated with DMSO, 3 μmol/L IK-175, or 3 μmol/L ITE. Media was refreshed every 2-3 days. On Day 11, cells were stimulated with Cell Stimulation Cocktail for 5 hours and stained for intracellular cytokines (CD4, IL17A, IL22). Samples were run on a BD LSR Fortessa flow cytometer and analyzed in FlowJo software. [2]
- Cell proliferation assays (human and murine): Human cancer cell lines (100 lines) were treated with nine concentrations of IK-175 (1 nmol/L to 10 μmol/L) for 3 days. Murine cell lines CT26 and B16-IDO1 were treated with six concentrations of IK-175 (33 nmol/L to 10 μmol/L) for 3 days. Cell viability was measured using CellTiter-Glo. [2]
- Flow cytometry for immune cell analysis: CT26 tumors and tumor-draining lymph nodes were harvested from mice treated with IK-175 (25 mg/kg, oral, once daily for 7 days). Cells were stained with surface and intracellular antibodies (CD45, CD3, CD4, CD8, IL2, IFNγ, TNFα, CD11b, F4/80, Ly6G, CD11c, Ly6C, CD206, MHCII) and Fixable Live/Dead. Samples were run on a BD LSR Fortessa flow cytometer and analyzed with FlowJo software. [2]

Animal/Disease Models: balb/c (Bagg ALBino) mouse (mouse colorectal cancer model CT26) [1]
Doses: 10 mg/kg (combined with anti-PD-1)
Route of Administration: Po; one time/day for 3 weeks
Experimental Results: obvious Inhibit tumor growth.

---

- Mouse PK study: Female Balb/c mice were administered IK-175 at 3 mg/kg either intravenously (5% DMSO, 75% PEG400, 20% water) or orally (30% PEG400, 10% Solutol, 60% water). Blood was collected over a time course and plasma levels measured by LC/MS. [2]
- Mouse pharmacodynamic study: Female Balb/c mice were orally administered VAG539 (30 mg/kg, AHR agonist prodrug) along with IK-175 (5, 10, or 25 mg/kg) in 0.5% methyl cellulose. Liver and spleen were harvested at 4 and 10 hours after dosing. Cyp1a1 RNA expression was measured by qRT-PCR. [2]
- B16-IDO1 efficacy study: Female C57BL/6 mice were implanted intradermally with B16-IDO1 cells (2 × 10⁵). Seven days post-implant, mice were randomized by tumor volume (average 30 mm³) and body weight. IK-175 (25 mg/kg in 0.5% methyl cellulose) was administered orally once daily. Anti-PD-1 antibody (10 mg/kg, clone RPM1-14) or rat IgG was injected intraperitoneally every 3 days for 5 doses. Tumors were measured three times per week. [2]
- CT26 efficacy study: Female Balb/c mice were implanted subcutaneously with CT26 cells (5 × 10⁵) in the right flank. Four days post-implant, mice were randomized by tumor volume (average 40 mm³) and body weight. IK-175 (25 mg/kg in 0.5% methyl cellulose) was administered orally once daily. Anti-PD-1 antibody (10 mg/kg) was injected intraperitoneally twice weekly for 5 doses starting on day 4. [2]
- Liposomal doxorubicin combination study (CT26): CT26 tumor-bearing mice were treated with liposomal doxorubicin (1 mg/kg, i.v., once weekly) and/or IK-175 (25 mg/kg, oral, once daily). Treatment started 7 days post-implant. Tumors were measured three times per week. [2]
- Phase I clinical trial: This was a first-in-human, open-label, multicenter, dose-escalation and expansion study (NCT04200963). IK-175 was administered orally in a fed state at doses from 200 mg to 1,600 mg daily (doses above 1,200 mg were given every 12 hours). In the combination arm, nivolumab 480 mg was administered intravenously every 4 weeks. The study enrolled 78 patients with advanced solid tumors (single-agent: n=14; combination: n=43). Primary endpoints were safety, tolerability, and determination of recommended phase 2 dose. Secondary endpoints included pharmacokinetics, pharmacodynamics, and preliminary antitumor activity. [3]

- Mouse PK: In Balb/c mice, IK-175 (3 mg/kg) showed oral bioavailability of approximately 50% with an elimination half-life of approximately 7 hours. [2]
- Rat PK: In Sprague-Dawley rats, IK-175 displayed good oral exposure over 24 hours. [2]
- Cynomolgus monkey PK: In cynomolgus monkeys, IK-175 displayed good oral exposure over 24 hours. [2]
- Human PK (Phase I): Following single and multiple dosing of IK-175 over 200-1,200 mg, median tmax ranged from 4 to 8 hours, with moderate to high variability (geometric CV% >30%). Half-life ranged from 4 to 6 hours across all dose levels. Cmax and AUC exhibited nonlinear pharmacokinetics (less than dose-proportional), with a plateau starting at 800 mg. Two active metabolites were detected; one metabolite approached equivalent exposures of IK-175 at the highest dose levels at steady state. IK-175 exposure was generally lower when administered with nivolumab than alone. The twice-daily dosing regimen (800 mg BID) showed similar Cmax and AUC as the daily regimen. [3]

- In the mouse PD study, no body weight loss was observed with IK-175 treatment at doses up to 25 mg/kg. [2]
- In B16-IDO1 and CT26 efficacy studies, no body weight loss was observed with IK-175 alone or in combination with anti-PD-1 or liposomal doxorubicin. [2]
- In the Phase I clinical trial, IK-175 was well tolerated. No dose-limiting toxicities were observed. No treatment-related adverse events with an outcome of death were reported. Immune-related adverse events (rash, proteinuria, adrenal insufficiency, pneumonitis, arthritis, supraventricular tachycardia) were infrequent (9% of patients overall) and manageable. The most common reasons for treatment discontinuation were progressive disease (48.7%) and clinical progression (29.5%); only 9% discontinued due to adverse events. [3]

[1]. Ahr inhibitors and uses thereof. WO2021142180A1.

[2]. Discovery and Characterization of a Novel Aryl Hydrocarbon Receptor Inhibitor, IK-175, and Its Inhibitory Activity on Tumor Immune Suppression. Mol Cancer Ther. 2022 Aug 2;21(8):1261-1272.

[3]. IK-175, an Oral Aryl Hydrocarbon Receptor Inhibitor, Alone and with Nivolumab in Patients with Advanced Solid Tumors and Urothelial Carcinoma. Clin Cancer Res. 2026 Jan 6;32(1):94-105

- In the mouse PD study, no body weight loss was observed with IK-175 treatment at doses up to 25 mg/kg. [2]
- In B16-IDO1 and CT26 efficacy studies, no body weight loss was observed with IK-175 alone or in combination with anti-PD-1 or liposomal doxorubicin. [2]
- In the Phase I clinical trial, IK-175 was well tolerated. No dose-limiting toxicities were observed. No treatment-related adverse events with an outcome of death were reported. Immune-related adverse events (rash, proteinuria, adrenal insufficiency, pneumonitis, arthritis, supraventricular tachycardia) were infrequent (9% of patients overall) and manageable. The most common reasons for treatment discontinuation were progressive disease (48.7%) and clinical progression (29.5%); only 9% discontinued due to adverse events. [3]

C25H27CL3FN7

550.886184930801

549.137

2247953-39-3

AHR antagonist 5 free base;2247950-42-9;AHR antagonist 5 hemimaleate

146047150

Yellow to orange solid powder

5

6

4

36

679

1

Cl.Cl.Cl.FC1=CN=CC(=C1)C1=NC2=C(C=NN2C(=N1)N[C@@H]1CCC2=C(C3C=CC=CC=3N2)C1)C(C)C

SRGGKFXUJGRKIJ-NMRXSTGRSA-N

InChI=1S/C25H24FN7.3ClH/c1-14(2)20-13-28-33-24(20)31-23(15-9-16(26)12-27-11-15)32-25(33)29-17-7-8-22-19(10-17)18-5-3-4-6-21(18)30-22;;;/h3-6,9,11-14,17,30H,7-8,10H2,1-2H3,(H,29,31,32);3*1H/t17-;;;/m1.../s1

(3R)-N-[2-(5-fluoropyridin-3-yl)-8-propan-2-ylpyrazolo[1,5-a][1,3,5]triazin-4-yl]-2,3,4,9-tetrahydro-1H-carbazol-3-amine;trihydrochloride

AHR antagonist-5 AHR antagonist 5

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)

DMSO : ~100 mg/mL (~181.52 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.54 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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.

---

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