Preclinical Pharmacological and Toxicological Evaluation of SB5794, a Novel Aryl Hydrocarbon Receptor Modulator on the Kynurenine–AhR Axis

Abstract

Conventional aryl hydrocarbon receptor (AhR) antagonists, which play a critical role in modulating tumor immune evasion, have shown limited clinical translation due to poor solubility, restricted systemic exposure, and dose-limiting toxicities. To overcome these limitations, we developed SB5794, a phosphate prodrug of the potent AhR antagonist SB2617, designed to improve aqueous solubility and pharmacokinetic properties. SB5794 exhibited markedly enhanced solubility and achieved more than six-fold higher systemic exposure in mice compared with SB2617, while fully retaining its in vitro AhR antagonistic activity. In syngeneic tumor models, SB5794 significantly inhibited tumor growth, and its combination with anti–PD-1 therapy further enhanced antitumor efficacy. However, repeated-dose studies revealed dose-dependent histopathological changes in the gastrointestinal tract, liver, and immune organs. Collectively, these findings demonstrate that SB5794 possesses improved drug-like properties and strong immunomodulatory activity, supporting its potential as a next-generation AhR-targeted immunotherapeutic candidate.

INTRODUCTION

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor belonging to the basic helix–loop–helix Per-Arnt-Sim (bHLH–PAS) family that regulates a wide range of biological processes in response to both endogenous metabolites and xenobiotics (Beischlag et al., 2008; Beischlag and Perdew, 2005; Denison and Nagy, 2003). Upon ligand binding, AhR translocates into the nucleus, forms a heterodimer with the AhR nuclear translocator (ARNT), and binds to xenobiotic response elements (XREs) to regulate gene transcription (Stockinger et al., 2014). Beyond its canonical role in xenobiotic metabolism, AhR signaling has emerged as a critical regulator of immune regulation, tumor progression, and host–microbe interactions (Gutierrez-Vazquez and Quintana, 2018; Murray et al., 2014; Powell and Ghotbaddini, 2014; Rothhammer and Quintana, 2019; Safe et al., 2013; Vogel and Haarmann-Stemmann, 2017).

Among the endogenous ligands, kynurenine (Kyn), a tryptophan metabolite produced by indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO2), is a well-characterized AhR activator (Hubbard et al., 2015; Mezrich et al., 2010; Opitz et al., 2011). Elevated Kyn levels and an increased Kyn/Trp ratio are associated with poor clinical outcomes and resistance to immune checkpoint inhibitors (Li et al., 2019; Long et al., 2019). Mechanistically, AhR activation promotes regulatory T-cell (Treg) differentiation, suppresses effector T-cell responses, and induces PD-1 expression in CD8⁺ T cells (Gandhi et al., 2010; Liu et al., 2018; Nguyen et al., 2010; Quintana et al., 2008). Consequently, the Kyn–AhR (Kynurenine-aryl hydrocarbon receptor) axis plays a pivotal role in tumor-associated immune suppression and represents an attractive therapeutic target (Platten et al., 2019) (Fig. 1).

Fig. 1.

Proposed mechanism of action of SB5794 targeting the kynurenine–AhR axis. Tryptophan is catabolized by IDO1 and TDO2 into kynurenine (Kyn), an endogenous ligand for the aryl hydrocarbon receptor (AhR). AhR activation in immune cells promotes regulatory T-cell (Treg) and M2 macrophage differentiation, suppresses effector CD8⁺ T-cell activity, and increases PD-1 expression, leading to immune evasion. SB5794, a phosphate prodrug of SB2617, inhibits Kyn-induced AhR signaling, restoring antitumor immune signaling through AhR inhibition. This blockade reprograms the tumor microenvironment by downregulating suppressive cells and enhancing stimulatory populations such as monocytes, M1 macrophages, and dendritic cells (DCs). These immunologic changes reactivate cytotoxic T-cell function and contribute to antitumor efficacy, particularly in combination with anti–PD-1 therapy.

Despite this therapeutic potential, the clinical translation of AhR antagonists has been hindered by poor aqueous solubility, low oral bioavailability, and dose-limiting toxicities, as observed with BAY964 and IK-175 (ClinicalTrials.gov, 2019a, 2019b; Ikena Oncology, 2022; Kober et al., 2023). These pharmacokinetic and safety limitations have restricted therapeutic efficacy and prevented progression beyond early-phase trials. To overcome these unmet needs, we developed SB2617, a potent AhR antagonist, and designed its phosphate prodrug, SB5794, to improve solubility and systemic exposure while maintaining full biological activity (Fig. 2).

Fig. 2.

Chemical structures of SB2617 and SB5794. SB2617 was identified as a potent AhR antagonist but exhibited poor solubility and limited systemic exposure. To overcome these limitations, the phosphate prodrug SB5794 was designed, showing markedly improved solubility, oral bioavailability, and in vivo exposure compared with the parent compound.

Our medicinal chemistry efforts focused on scaffold optimization, bioisosteric modification, and prodrug design (Sun et al., 2020) to address these limitations. Phosphate prodrugs have been proven effective in enhancing solubility and pharmacokinetic performance without compromising pharmacodynamic integrity (Rautio et al., 2018; Testa, 2009; Testa and Mayer, 2003; Xu et al., 2023; Zhang et al., 2023). Accordingly, SB5794 was designed to improve oral bioavailability and achieve pharmacologically relevant plasma concentrations while preserving potent AhR antagonism.

In this study, we report the physicochemical, pharmacokinetic, and pharmacological properties of SB5794 in comparison with SB2617, as well as its in vivo efficacy and toxicity profiles. These findings demonstrate the feasibility of a prodrug-based approach to overcome the druggability limitations of AhR antagonists and support further development of SB5794 as a next-generation immunomodulatory agent targeting the Kyn–AhR signaling axis.

MATERIALS AND METHODS

Chemicals

SB2617, a pyrido[3,4-d]pyrimidin-4(3H)-one derivative, and its phosphate prodrug SB5794 were synthesized according to the procedure described in WO2021/210970 A1 (Song et al., 2021). For in vitro assays, compounds were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA) and subsequently diluted with cell culture medium to achieve the desired concentrations.

Animals

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Dong-A ST Research Institute (protocol Nos. I-2201006, I-2201009, I-2203054, I-2208115, I-2209126, I-2210140, I-2211146, I-2211147, I-2211156) and conducted according to ARRIVE guidelines. Male BALB/c (7-8 weeks) mice were used for pharmacokinetic studies, male C57BL/6 (6-8 weeks) for efficacy studies (MC38 model), and male ICR (7-8 weeks) for repeated-dose studies. Animals were obtained from Orient Bio (Korea), acclimated ≥7 days, and maintained under SPF conditions (22 ± 2°C; 50 ± 10% humidity; 12-h light/dark cycle) with ad libitum access to food and water.

Randomization and blinding

For efficacy studies, mice were block-randomized based on baseline tumor volume. Tumor measurements were performed by investigators blinded to treatment allocation. Exclusion criteria included tumor ulceration, humane endpoint achievement, or dosing errors.

Cell lines and culture conditions

HepG2 (human hepatocellular carcinoma) and CT26 (mouse colon carcinoma) cells were maintained in DMEM (Gibco, USA) supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a humidified 5% CO₂. All cell lines were verified to be mycoplasma-free before use.

In vitro assays

AhR reporter gene assay: AhR antagonistic activity was assessed using a luciferase reporter assay in HepG2-XRE cells. Cells (2×10⁴/well) were seeded in 96-well plates and incubated overnight before treatment with kynurenine (100 µM) in the presence or absence of test compounds for 24 h. Luciferase activity was measured in triplicate using the Dual-Luciferase Reporter Assay System (Promega, USA) and normalized to Renilla activity.

Pharmacokinetics (PK) studies

Pharmacokinetic studies were performed in BALB/c mice. Compounds were formulated in 10% DMSO, 40% PEG 400, and 50% saline for i.v. administration or in 0.5% methylcellulose for p.o. dosing. Mice (n=3/time point) received SB2617 (5 mg/kg, i.v.; 5 mg/kg, p.o.) or SB5794 (5 mg/kg, p.o.). Blood samples were collected at 0.25-24 h, and plasma concentrations were quantified by LC–MS/MS. Pharmacokinetic parameters (C_max, T_max, t½, AUC) were determined by noncompartmental analysis using Phoenix WinNonlin (Certara, USA).

In vivo efficacy studies

MC38 cells (5×10⁵) were implanted subcutaneously into male C57BL/6 mice. When tumors reached 100-150 mm³, mice were randomized and treated with SB5794 (10 mg/kg, p.o., daily), vehicle, anti-PD-1 (10 mg/kg, i.p., twice weekly), or the combination. Tumor volume (V=L×W²/2) and body weight were recorded every 2-3 days.

Toxicity studies

Repeated-dose toxicity was evaluated in male ICR mice (vehicle: n=6; SB5794 dose groups: n=9 [6 main+3 TK]) administered SB5794 orally once daily at 30, 100, or 300 mg/kg for 14 days, conducted with reference to the principles described in ICH M3(R2) for exploratory repeated-dose toxicity evaluation and under GLP-compliant conditions. Vehicle controls received 0.1% kolliphor EL. Clinical signs, body weight, food consumption, and gross appearance were monitored throughout the study. At study termination, blood and a comprehensive panel of organs (stomach, small and large intestine, pancreas, liver, spleen, thymus, kidney, lung, heart, adrenal gland, and other major tissues) were collected for hematological, clinical chemistry, organ weight, and histopathological (H&E) evaluations. This exploratory study was carried out in a single sex (male) in line with our institutional early-stage toxicology platform, which is based on an extensive historical control database for male ICR mice. For oral toxicity studies, 0.1% kolliphor EL was selected as the vehicle to enable safe repeated high-dose administration, whereas 0.5% methylcellulose was used only in the single-dose PK study; this difference in vehicle selection did not affect the interpretation of systemic exposure or safety margins.

Comparative toxicity study with BAY964

A comparative repeated-dose toxicity study was conducted to contextualize the safety profile of SB5794 relative to BAY964. Male ICR mice received BAY964 orally once daily at 30, 100, 300, or 1000 mg/kg for 14 days under GLP-compliant conditions, following the same study design used for SB5794. Evaluated parameters included clinical signs, body weight, food and water consumption, hematology, serum biochemistry, organ weight, and full histopathology. Toxicokinetic samples were collected on Days 1 and 14. Notably, the 30 mg/kg dose of BAY964 was evaluated only in a single-dose toxicokinetic study and was not included in the 14-day repeated-dose toxicity assessment.

Statistical analysis

Data were analyzed using GraphPad Prism 9.0. Tumor growth was analyzed by mixed-effects model (REML) with Geisser–Greenhouse correction and Tukey’s test. Tumor growth inhibition (TGI) was calculated as 1 − (ΔT/ΔC) on day 28. Student’s t-test was used for pairwise comparisons. Normality and variance were assessed by Shapiro–Wilk and Levene’s tests. Data are presented as mean ± SEM, and statistical significance was defined as p<0.05.

RESULTS

Design and identification of AhR antagonists

A library of pyrido[3,4-d]pyrimidin-4(3H)-one derivatives was synthesized and screened for AhR antagonistic activity. Among these, SB2617 emerged as the most potent compound (IC₅₀=1.03 nM), featuring a 4-chlorophenyl substituent at the 6-position, a pyridin-3-yl group at the 8-position, and a (1R,4R)-4-hydroxycyclohexyl group at the 3-position. However, its poor aqueous solubility (<0.1 mg/mL) led to limited oral exposure. To overcome this, a phosphate prodrug, designated SB5794, was synthesized. SB5794 exhibited a more than 100-fold increase in solubility while maintaining in vitro potency (IC₅₀=50.03 nM) after bioconversion to SB2617.

In vitro pharmacological characterization

Using an XRE-driven luciferase reporter assay in HepG2 cells, both SB2617 and SB5794 demonstrated potent inhibition of kynurenine-induced AhR activation. The comparable potency of SB5794 (IC₅₀=0.15 µM) confirmed efficient enzymatic conversion to the active SB2617 under in vitro conditions.

Pharmacokinetic properties

Pharmacokinetic (PK) analysis in BALB/c mice revealed markedly improved systemic exposure for SB5794 compared with SB2617 (Table 1). Oral administration of SB2617 resulted in poor bioavailability (<5%), while SB5794 achieved an oral bioavailability of approximately 35%. LC–MS/MS confirmed rapid in vivo conversion of SB5794 to SB2617 within 1 h post-dose. This conversion occurs through enzymatic dephosphorylation mediated by endogenous phosphatases, including alkaline phosphatase. For SB5794, systemic exposure was quantified as the plasma concentration of the active parent compound SB2617 following in vivo dephosphorylation. Systemic exposure (AUC) increased more than six-fold compared with oral SB2617. Dose-dependent pharmacokinetics were confirmed in a single-dose toxicokinetic (TK) study (100-1000 mg/kg), showing proportional increases in C_max of SB2617, as shown in Table 2. Corresponding plasma concentration–time profiles are presented in Fig. 3.

Table 1.

Pharmacokinetic parameters of SB2617 and SB5794 in BALB/c mice^a,b

PK parameters	SB2617 (i.v., 5 mg/kg)	SB2617 (p.o., 5 mg/kg)	SB5794 (i.v., 5 mg/kg)	SB5794 (p.o., 5 mg/kg)
Terminal t1/2 (h)	13.05	-	16.4	17.6
Tmax (h)c	-	17.3	-	6.00
Cmax (ng/mL)	-	365	-	534
AUC0–last (ng·h/mL)	11325	6730	14457	8710
AUCinf (ng·h/mL)	15719	-	22255	15203
CL (mL/min/kg)	5.4	-	3.8	-
Vd (L/kg)	5.75	-	5.17	-
Bioavailability (%)d	-	59.4	-	60.3
Relative % to SB2617	-	-	107.7	109.2

ᵃMice (n=3) received SB2617 or SB5794 (5 mg/kg) by i.v. or p.o. routes. SB5794 exhibited markedly improved pharmacokinetic properties compared with SB2617, including higher C_max, AUC, and oral bioavailability. ᵇData are expressed as mean values (n=3). ᶜT_max is given as median. ᵈBioavailability (F%) was calculated relative to SB2617 p.o. dosing. ‘Relative % to SB2617’ was calculated using parent-equivalent exposure, defined as: (AUC_0–last of SB5794×MW_SB2617/MW_SB5794)/(AUC_0–last of SB2617)×100.

This metric reflects systemic exposure normalized for the molecular-weight difference between the phosphate prodrug (SB5794) and the parent compound (SB2617).

Table 2.

Pharmacokinetic parameters of SB5794 and SB2617 in BALB/c mice (single-dose TK study)^a,b

PK parameters	SB5794 (p.o.)				SB2617 (p.o.)
	100 mg/kg	300 mg/kg	1000 mg/kg		100 mg/kg	300 mg/kg
Terminal t1/2 (h)	-	-	-		29.70	31.5
Tmax (h)c	11.3	17.3	26.0		7.3	14.7
Cmax (ng/mL)	12600	23733	51500		1502	2659
AUC0-24 (ng·h/mL)	261982	509222	1100298		32107	57542
AUC0-last (ng·h/mL)	325175	641778	1396410		-	-
AUC vs SB2617 10 mg/kg	15.0	29.2	63.2		1.8	3.4

ᵃMice (n=3 per group) received SB5794 (100, 300, or 1000 mg/kg) or SB2617 (100 or 300 mg/kg) orally. ᵇSB5794 demonstrated significantly improved C_max and AUC compared with SB2617. ᶜT_max is given as median.

Fig. 3.

Plasma concentration–time profiles of SB2617 and SB5794 in BALB/c mice. Mice (n=3) were administered SB2617 or SB5794 (5 mg_∙kg^–¹) via i.v. or p.o. routes. Plasma levels of SB2617 were determined by LC–MS/MS. Oral administration of SB5794 achieved significantly higher systemic exposure and improved bioavailability compared with SB2617.

In vivo antitumor efficacy

As summarized in Table 3 and Fig. 4A, oral administration of SB5794 (10 mg/kg, once daily) significantly inhibited tumor growth in the MC38 syngeneic colon carcinoma model using male C57BL/6 mice. On day 28, tumor growth inhibition (TGI) reached 38.4% for SB5794 monotherapy and 54.2% for the SB5794 plus anti–PD-1 combination group, with no complete responses observed. Body weight remained stable across all treatment groups throughout the study (Fig. 4B), and no significant differences were detected (p>0.05).

Table 3.

Antitumor efficacy of SB5794 in the MC38 syngeneic tumor model^a,b

Treatment	No. of animals	CR	TGI (%) Day 28
Control	9	0/9	0.0
αPD-1	9	0/9	35.0
G5 (SB5794, 10 mpk)	9	0/8	38.4
G6 (SB5794, 10 mpk+αPD1)	9	0/9	54.2

ᵃData are expressed as mean values (n=9). CR, complete response; TGI, tumor growth inhibition. ᵇMice were treated with vehicle, anti–PD-1 antibody, SB5794 (10 mg/kg), or the combination of SB5794 with anti–PD-1. Tumor growth inhibition (TGI) was calculated on day 28.

Fig. 4.

Antitumor efficacy of SB5794 in the MC38 syngeneic tumor model. (A) Tumor growth curves for MC38-bearing mice treated with vehicle, anti–PD-1 antibody (αPD-1), SB5794, or the combination of SB5794 with αPD-1. SB5794 monotherapy significantly inhibited tumor growth, and the combination further enhanced efficacy. (B) Body weight changes during the study period. No significant body weight loss was observed in any treatment group, indicating that SB5794 was well tolerated.

Toxicity profile

SB5794 was evaluated for safety and tolerability in short-term efficacy studies and a 2-week repeated-dose toxicity study conducted under GLP conditions (OECD Guideline 407). Male ICR mice (n=9/group) received SB5794 orally at doses of 30, 100, or 300 mg/kg in the 2-week repeated-dose toxicity study. No notable clinical signs or body-weight changes were observed at the pharmacologically active dose in the short-term studies. In the repeated-dose study, SB5794 induced clear dose-dependent toxicities. At 30 mg/kg, minimal findings were observed, limited to pancreatic acinar cell vacuolation and mild gastric epithelial changes. At 100 mg/kg, histopathological lesions became more evident, including mononuclear cell infiltration and epithelial vacuolation in the gastrointestinal tract, hepatic apoptosis with increased extramedullary hematopoiesis, and splenic hematopoietic changes. At 300 mg/kg, more severe and widespread toxicities occurred, such as diffuse GI mucosal atrophy, pancreatic necrosis, hepatocellular apoptosis/necrosis with glycogen depletion, marked thymic atrophy, and cardiac/perivascular mononuclear infiltration, which led to early terminations in this group due to severe toxicity. Body-weight changes were minimal at 30 mg/kg and comparable to vehicle controls, indicating no treatment-related effect. At 100 mg/kg, a transient decrease (~12% at day 6) was observed but recovered by study end. In contrast, the marked 21% reduction at 300 mg/kg was consistent with systemic toxicity requiring early termination. No no-observed-adverse-effect level (NOAEL) was identified within the tested dose range. Comprehensive toxicological findings (clinical observations, hematology, serum biochemistry, and histopathology) are summarized in Table 4.

Table 4.

Toxicological findings of SB5794 in a 2-week repeated-dose toxicity study in mice^a,b

Parameter	Vehicle control	SB5794 (30 mg/kg)	SB5794 (100 mg/kg)	SB5794 (300 mg/kg)
TK AUClast (ng·h/mL)	-	[D1] 61,924 (3.6×) [D14] 81,760 (4.7×)	[D1] 237,032 (13.6×) [D14] 532,458 (30.6×)	[D1] 386,380 (22.2×)
BW (change, %)	5.7%	9.5%	0.4% (↓12% at D6, recovered)	↓21% at D9
Food/Water intake	-	-	↓ at D3, D7	↓ at D3 and D7
Mortality	0/6	0/9	0/9	2/9 FDC, 7/9 moribund → terminated at D9
Clinical signs	-	-	-	Hypoactivity, piloerection (D3-9)
Organ weight	-	-	Liver absolute/relative ↑ (141%, 149%)	-
Hematology	-	-	↑ Mono (%), Retic (%)	-
Biochemistry	-	-	↑ Crea, GOT, GPT, LDH ↓ HDL-chol, ↑ T-chol, ↑ TBIL, ↑ TG	-
Histopathology	-	-	Lesions in liver, spleen, lung, kidney	Lesions in thymus, stomach, kidney, spleen, lung

Comparative toxicity with BAY964

A comparative repeated-dose toxicity study was conducted with BAY964, a clinical-stage AhR antagonist, to contextualize the safety profile of SB5794. As summarized in Table 5, SB5794 and BAY964 showed distinct organ-level toxicity profiles across comparable dose ranges. For BAY964, no repeated-dose toxicity findings were available at 30 mg/kg because this dose was included only in a single-dose TK assessment and not in the 14-day study. Target organs affected by SB5794 included the stomach, liver, spleen, kidney, and lung, whereas BAY964 primarily affected the liver, spleen, gastrointestinal tract, and kidney. A definitive NOAEL could not be identified for SB5794 within the tested dose range, because even the lowest dose (30 mg/kg) produced treatment-related histopathological findings.

Table 5.

Comparative in vivo toxicological findings of SB5794 and BAY964 in mice^a,b

Dose group	SB5794 (SB2617-based)	BAY964 (competitor)
30 mg/kg	[3.6×; D1, 4.7×; D14] - Stomach, intestine, spleen	No repeated-dose toxicity data available (TK only)
100 mg/kg	[13.6×; D1, 30.6×; D14] - Liver ↑, Mono/Retic ↑, clinical chem. changes	[1.9×] - MCHC ↓, TG ↓, liver, spleen
300 mg/kg	[22.2×; D1] D9 FDC, overall termination; lesions in liver, heart, spleen	[3.6×] - liver, spleen; hematological changes
1000 mg/kg	-	[7.0×] - gastrointestinal, liver, spleen, kidney lesions
Target organ	Stomach (with contents), liver, heart, spleen, lung, kidney	Liver, spleen, GI tract, kidney
Estimated NOAEL	Not identified	<100 mg/kg

ᵃD1 and D14 indicate the day of sampling after dosing. FDC, found dead condition; NOAEL, no-observed-adverse-effect level. ᵇMice were treated with SB5794 or BAY964 at various doses for a 2-week repeated-dose toxicity study. Parameters evaluated included body weight, food/water intake, clinical signs, hematology, serum biochemistry, and histopathology. Target organs affected and the estimated NOAEL are indicated.

For BAY964, the estimated NOAEL was approximately 100 mg/kg.

In vitro safety evaluation

Potential cardiotoxicity and genotoxicity were evaluated in vitro. As summarized in Table 6, neither SB5794 nor BAY964 inhibited hERG channel activity (IC₅₀>10 µM), and both were negative in Ames and chromosomal aberration assays, indicating minimal risk of cardiotoxicity or genotoxicity.

Table 6.

In vitro safety profile of SB5794 compared with competitor compound BAY964^a,b

Assay	SB5794 (SB2617-based)	BAY964 (competitor)
hERG IC50 (µM)	>10 µM	>10 µM
Ames test	Negative	-
Chromosomal aberration (CA)	Negative	-

DISCUSSION

In this study, we characterized the preclinical pharmacological and toxicological profiles of SB5794, a phosphate prodrug of SB2617, which was designed to overcome the physicochemical and pharmacokinetic limitations that have restricted the clinical progress of earlier aryl hydrocarbon receptor (AhR) antagonists. The kynurenine–AhR signaling axis plays a central role in tumor-induced immune suppression by promoting regulatory T-cell differentiation, upregulating PD-1 expression, and attenuating effector T-cell responses. Accordingly, inhibition of AhR signaling represents a promising immuno-oncology approach. However, the clinical utility of first-generation AhR antagonists, such as BAY964 and IK-175, has been hindered by poor aqueous solubility, low bioavailability, and dose-limiting toxicity. To address these issues, SB5794 was rationally designed as a phosphate prodrug of SB2617, a potent but poorly soluble AhR inhibitor, with the goal of improving solubility, systemic exposure, and overall druggability while retaining full AhR antagonistic activity.

SB5794 achieved more than a 100-fold increase in solubility and over six-fold higher systemic exposure compared with SB2617 in mice. Pharmacokinetic profiling confirmed rapid enzymatic conversion of SB5794 to SB2617 within one hour following oral administration, resulting in a bioavailability of approximately 35% versus less than 5% for the parent compound. These findings indicate that the phosphate modification enhanced oral absorption while preserving metabolic stability, consistent with the established performance of other clinically validated phosphate prodrugs such as fosphenytoin and fosaprepitant. Thus, SB5794 exemplifies how prodrug engineering can effectively improve the oral pharmacokinetic properties of hydrophobic small molecules targeting AhR.

Pharmacodynamic outcomes were closely aligned with the improved pharmacokinetics of SB5794. In vitro, SB5794 demonstrated complete retention of AhR inhibitory potency in HepG2-XRE reporter assays, confirming efficient dephosphorylation to the active SB2617. In vivo, SB5794 produced significant antitumor activity in the MC38 syngeneic colon carcinoma model, showing 38% tumor growth inhibition (TGI) as monotherapy. When combined with anti–PD-1 antibody, SB5794 yielded an enhanced TGI of 54% without significant changes in body weight or overt toxicity. These findings suggest additive or potentially synergistic efficacy through complementary mechanisms. Given that AhR signaling promotes PD-1 expression and contributes to T-cell exhaustion, dual blockade of AhR and PD-1 may effectively restore cytotoxic T-cell function and augment antitumor immunity. Therefore, combination therapy involving AhR inhibition and checkpoint blockade represents a rational strategy for further clinical exploration.

SB5794 was generally well tolerated at pharmacologically active doses, although dose-dependent toxicities were observed at ≥100 mg/kg. Histopathological analyses revealed lesions primarily in the gastrointestinal tract, liver, spleen, and immune-related organs. These findings are consistent with either on-target or metabolite-related effects, as AhR is known to be expressed in hepatocytes and intestinal epithelial cells, where it regulates xenobiotic metabolism and mucosal immune balance. The lesions at high doses may result from local intestinal exposure or systemic metabolic stress related to phosphate cleavage and hepatic conversion. Importantly, no adverse clinical signs were observed within the therapeutic range (≤30 mg/kg), suggesting a favorable safety margin for continued development. However, the absence of a clearly defined no-observed-adverse-effect level (NOAEL) indicates that extended non-rodent toxicity studies will be necessary to more precisely determine the therapeutic window.

We also acknowledge that the repeated-dose toxicity study was conducted only in male ICR mice, and future longer-term regulatory toxicology studies will include both sexes to fully evaluate potential sex-dependent differences.

When compared with BAY964, a clinical-stage AhR antagonist, SB5794 demonstrated higher systemic exposure and broader tissue distribution, likely due to improved solubility and bioavailability. Although such increased exposure contributed to superior antitumor efficacy, it may also account for the wider organ involvement at higher doses. These results highlight a critical consideration in prodrug design: while greater systemic exposure enhances pharmacological activity, it necessitates careful dose optimization to prevent off-target or tissue-specific toxicity. Importantly, in vitro safety evaluations showed no hERG channel inhibition (IC₅₀>10 μM) and negative results in Ames and chromosomal aberration assays, suggesting minimal risk of cardiotoxicity or genotoxicity. Therefore, the observed toxicities likely stem from pharmacologic and metabolic effects rather than intrinsic chemical liabilities.

In conclusion, SB5794 represents a next-generation AhR-targeted immunomodulator that successfully integrates enhanced solubility, favorable pharmacokinetics, potent target inhibition, and strong in vivo efficacy. The consistent pharmacokinetic–pharmacodynamic (PK–PD) relationships observed between plasma exposure, AhR pathway inhibition, and tumor suppression establish a robust mechanistic connection from molecular engagement to therapeutic effect. Furthermore, the superior performance of SB5794 in combination with anti–PD-1 underscores a promising immunoregulatory mechanism capable of reprogramming the kynurenine-driven immunosuppressive tumor microenvironment.

Future studies should focus on elucidating tissue-level target occupancy, mechanisms of resistance, and the identification of translational biomarkers such as plasma kynurenine concentrations and AhR-responsive gene signatures. Taken together, SB5794 can be regarded as an orally bioavailable, mechanistically validated AhR-targeted immunotherapeutic that represents a significant advancement in the pharmacological modulation and translational development of the Kyn–AhR axis.