Indoleamine 2,3-dioxygenase 1 (IDO1) (IC50 = 1.1 nM)

When SKOV-3 and Jurkat clone E6-1 cells are treated with linrodostat (0.01-100 μM) for 72 hours, the proportion of viable cells is lower than when the control group is left untreated. With an IC50 of 6.3 μM, linrodostat likewise causes cell death at substantially lower concentrations[1].

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In support of clinical testing, BMS-986205 was evaluated preclinically. In vitro characteristics included potent inhibition of kynurenine (kyn) production in IDO1-HEK293 cells (IC50 = 1.1 nM) but not in TDO-HEK293 cells, sustained inhibition in IDO1 cell-based assays after washout, and single-digit nM potency in human tolerogenic MLR assays. [2]

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Linrodostat exhibited potent cellular activity, suppressing kynurenine production in HEK293 cells overexpressing human IDO1 and HeLa cells stimulated with IFNγ, with no activity against tryptophan 2,3-dioxygenase or murine indoleamine 2,3-dioxygenase 2 detected. Linrodostat restored T-cell proliferation in a mixed-lymphocyte reaction of T cells and allogeneic IDO1-expressing dendritic cells [3].

Based on preclinical data, a 150 mg QD human dose was projected to maximally inhibit IDO1. In the ongoing clinical study, 42 pts have been treated. All treatment-related adverse events were grade 1/2 except three grade 3 toxicities (autoimmune hepatitis [dose limiting; BMS-986205 100 mg/nivo 240 mg], rash, and asymptomatic hypophosphatemia). Day 14 individual trough concentrations began exceeding the human whole blood IC50 starting with 25 mg QD, and the IC90 starting with 50 mg QD; all pts treated at 200 mg QD exceeded the IC90. Serum kyn was substantially reduced at all doses (> 45% mean reduction at each dose), with > 60% mean reduction at the 100 and 200 mg QD doses. Importantly, intratumoral kyn was reduced up to 90% in evaluable paired pre- and on-treatment samples.

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Linrodostat PK/PD profile in human SKOV3 xenografts [3]
Next, we assessed PK/PD properties of linrodostat using SKOV3 cells in a mouse xenograft model. Dose-dependent PD activity of linrodostat was assessed by measuring serum and intratumoral kynurenine levels with dosing once a day at 5, 25, or 125 mg/kg (Supplementary Table S1; PK reported as AUC0–24, μmol/L × h). AUEC measurements for the percent kynurenine reduction in linrodostat-treated tumors were 60%, 63%, and 76% at 5, 25, and 125 mg/kg, respectively, demonstrating dose-dependent PD activity in tumors. Of the time points assessed, maximal PD effects (96% kynurenine reduction) were observed at 6 hours after treatment (Fig. 4A). Reduced kynurenine levels (≥ 30%) were maintained 24 hours after the last dose of 10 mg/kg linrodostat and 100 mg/kg epacadostat. Derived from these PK/PD analyses, the in vivo median IC50 of linrodostat was 3.4 nmol/L in serum from SKOV3 tumors, which was similar to the in vitro median IC50 of 9.4 nmol/L in human whole blood (Fig. 4B). We also examined the potency of another IDO1 inhibitor, epacadostat, and detected a median IC50 of 227- and 163 nmol/L potency in vivo and in vitro, respectively (Fig. 4C). These data demonstrate that linrodostat shows single-digit nanomolar potency.

IDO1 inhibition [3]
SKOV3 human ovarian cancer cells (ATCCs) were treated with media containing 1 mmol/L tryptophan, 50 μmol/L succinylacetone, and 50 ng/mL human IFNγ to induce IDO1 synthesis, and either vehicle (DMSO), Linrodostat , or epacadostat at 10, 50, or 100 nmol/L for 72 hours. Kynurenine levels were measured with the Ehrlich colorimetric assay. Colorimetric changes were detected with a plate reader (490 nm). Kynurenine concentrations were determined from kynurenine standard curves.

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Reversal of IDO1 inhibition [3]
SKOV3 cells were treated with 5 μg/mL cycloheximide to prevent further IDO1 synthesis and incubated with 50 μmol/L succinylacetone. Media were replaced with media containing 1 mmol/L tryptophan, 50 μmol/L succinylacetone, and 5 μg/mL cycloheximide, and duplicates were treated with vehicle (DMSO) or 10 μmol/L heme. Kynurenine levels were measured as described above at 0, 2, 4, 6, 8, and 10 hours.

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Aryl hydrocarbon receptor assay [3]
The Human AhR Reporter Assay System (96-well format) was used according to the manufacturer's instructions. Reporter cells dispensed into a 96-well plate were immediately dosed with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 1 or 10 nmol/L), kynurenine (1 or 10 μmol/L), or Linrodostat (1 or 10 μmol/L). After 22 to 24 hours' incubation, media were removed, and luciferase detection reagent (100 μL) was added. After approximately 5 minutes, luciferase activity was detected per well using a plate-reading luminometer.

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SKOV-3 IDO1 assay (cell plating, IDO1 induction, kynurenine determination) [1]
The cells were plated at 3 × 104 cells/well and allowed to attach overnight. The next day IFNγ was added to the cell culture at 100 ng/mL final concentration to induce IDO1 expression followed by 24 h incubation at 37°C and 5% CO2. A kynurenine standard curve was used to interpolate kynurenine concentrations in the test samples.

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Caspase 3/7 activation [1]
Caspase 3/7 activity was measured using Caspase 3/7 Glo reagent form Promega according to manufacturer's instructions.

Half maximal inhibitory concentration in cell-based assays [3]
All cell lines used in the study were routinely tested for mycoplasma, and passages were tracked per standard protocols. HEK293 cells overexpressing human IDO1, human TDO, murine Ido1, or murine Ido2 were seeded using RPMI phenol red–free media containing 10% fetal bovine serum (FBS). Linrodostat was prepared as previously described in Patent Cooperation Treaty Int Appl WO 2016073774 (example 19; refs. 32, 33). Linrodostat (100 nL; 10 μmol/L–10 pmol/L) was added, and cells were incubated for 20 hours at 37°C in 5% CO2. HeLa or M109 cells were seeded using RPMI phenol red–free media containing 10% FBS. Linrodostat (50 μL; 10 μmol/L–10 pmol/L) was added, and cells were incubated at 37°C for 2 hours. Recombinant human IFNγ (final concentration, 10 ng/mL) or recombinant murine IFNγ (final concentration, 5 ng/mL) was added to induce IDO1. Cells were incubated for 18 hours at 37°C in 5% CO2. Treatment was stopped, and IC50 values were calculated.

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Relative viability determination [1]
Cell viability was assessed using Cell-Titer Fluro (Promega) according to manufacturer's instructions. Relative cell viability was calculated based on the reading of the non-treated control. % Relative Viability = TestValue/Non-treatedControlValue × 100.

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Cell Viability Assay[1]
Cell Types: SKOV-3 and Jurkat clone E6-1 cells
Tested Concentrations: 0.01-100 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: decreased the number of viable cells compared with the non-treated control and induced cell death at much lower concentrations.

During dose escalation, patients (pts) with advanced cancers were treated in escalating cohorts with BMS-986205 (25-200 mg as of Jan 5, 2017) orally once daily (QD) for 2 wk, followed by BMS-986205 + nivo 240 mg IV every 2 wk. Objectives included safety (primary), PK, and PD. [2]

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Human SKOV3 xenograft tumor model [3]
Female nu/nu mice (7–12 weeks old; Envigo) received food and water ad libitum and were maintained in a controlled environment according to Association for Assessment and Accreditation of Laboratory Animal Care International regulations. SKOV3 cells were maintained in RPMI 1640 with 10% FBS and were harvested in the log growth phase (6 × 107 cells/mL in Hank's balanced salt solution), mixed 1:1 with Matrigel, and s.c. implanted into the mouse flank (0.1 mL or 3 × 106 cells/mouse). After implantation (15 days), tumor volumes were measured, and tumor-bearing mice were randomized (five animals/group). Linrodostat was formulated as a solution in the vehicle ethanol/polyethylene glycol (PEG) 400/propylene glycol/d-α-tocopheryl PEG 1000 succinate (volume ratio, 5:55:20:20). Each group was orally administered a vehicle, Linrodostat (5, 25, or 125 mg/kg once a day), or epacadostat (30 or 100 mg/kg twice daily), for 5 days. Tumor volumes were measured before tumors were snap frozen. Sera were harvested (day 5) at designated times after dosing to quantify kynurenine and compound levels.

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PK analyses [3]
PK parameters were determined by a noncompartmental model of analysis of plasma concentration versus time data using Phoenix 6.3.0.395. AUCs from time zero to last sampling time (AUC0-T) and time zero to infinity (AUCINF) were calculated using the linear-log trapezoidal method. The total plasma clearance (CLTp), steady-state volume of distribution (Vss), and apparent elimination half-life (t1/2) were estimated after IV administration. t1/2 estimations were calculated at ≥ 3 time points. The total blood clearance (CLTb) was calculated as the CLTp divided by the blood-to-plasma concentration ratio. The absolute oral bioavailability (F) was estimated as the ratio of dose-normalized AUCs following oral and IV doses.

Pharmacokinetic characteristics of linrostat [3] To extend these SKOV3 xenograft studies, we evaluated the pharmacokinetic parameters of linrostat after intravenous and oral administration in higher animals, including rats, dogs, and monkeys (Table 1). After intravenous administration, the hepatic blood flow (CLTp) of linrostat was comparable across species, with CLTp values of 27, 25, and 19 mL/min/kg in rats, dogs, and monkeys, respectively. CLTp ≤ 48% of the corresponding reported hepatic blood flow indicates low to moderate systemic clearance of linrostat. After intravenous administration, the steady-state volume of distribution (Vss) values in rats, dogs, and monkeys were 3.8, 5.7, and 4.1 L/kg, respectively, indicating extravascular distribution. The half-life (t1/2) of linrostat in rats, dogs, and monkeys were 3.9, 4.7, and 6.6 hours, respectively. After oral administration, the time to peak concentration (Tmax) in rats, dogs, and monkeys ranged from 0.5 to 1.7 hours. Linrostat, administered in solution, had absolute oral bioavailability of 64%, 39%, and 10% in rats, dogs, and monkeys, respectively. The rate of metabolism of linrostat by rat hepatocytes was higher than that in dogs, monkeys, and humans. Based on the in vitro-in vivo correlation of hepatocyte clearance in animals, moderate clearance in humans is expected. These data suggest an expected human half-life (t1/2) of 23 hours, supporting once-daily dosing in clinical settings. To extend these preclinical in vivo pharmacodynamic/pharmacokinetic (PD/PK) studies, we evaluated the pharmacodynamics of kynurenine in patients with advanced cancer treated with linrostat in combination with nivolumab. A significant reduction in mean serum kynurenine levels was detected at all evaluated linrostat doses, with a reduction of approximately 60% at once-daily doses of 100 mg and 200 mg (Figures 5A and B). Furthermore, tumor samples from 13 patients before and during treatment showed that, even with relatively high kynurenine levels before treatment, intratumoral kynurenine levels decreased in all dose groups after treatment (Figure 5C). These results indicate that linrostat in combination with nivolumab can significantly reduce serum and intratumoral kynurenine levels.

[1]. Cell based functional assays for IDO1 inhibitor screening and characterization. Oncotarget. 2018 Jul 20;9(56):30814-30820.

[2]. Abstract CT116: BMS-986205, an optimized indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor, is well tolerated with potent pharmacodynamic (PD) activity, alone and in combination with nivolumab (nivo) in advanced cancers in a phase 1/2a trial. AACR; Cancer Res. 2017; 77 (13 Suppl): Abstract nr CT116.

[3]. Preclinical Characterization of Linrodostat Mesylate, a Novel, Potent, and Selective Oral Indoleamine 2,3-Dioxygenase 1 Inhibitor. Mol Cancer Ther. 2021 Mar;20(3):467-476.

Linrodostat is being investigated in the clinical trial NCT03247283 (pharmacokinetic and metabolic study in healthy male subjects). Linrodostat is an orally administered indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor with potential immunomodulatory and antitumor activity. After administration, linrodostat specifically targets and binds to IDO1, a cytoplasmic enzyme responsible for oxidizing the amino acid tryptophan to kynurenine, an immunosuppressive metabolite. By inhibiting IDO1 and reducing kynurenine levels in tumor cells, BMS-986205 restores and promotes the proliferation and activation of various immune cells, including dendritic cells (DCs), natural killer (NK) cells, and T lymphocytes, and reduces tumor-associated regulatory T cells (Tregs). In many cancers, the immune system is suppressed, and activation of the immune system may induce a cytotoxic T lymphocyte (CTL) response against IDO1-expressing tumor cells, thereby inhibiting the growth of IDO1-expressing tumor cells. IDO1 is overexpressed in various tumor cell types and plays an important role in immunosuppression. Tryptophan depletion inhibits the proliferation and activation of T lymphocytes, thereby suppressing the immune system. Indoleamine 2,3-dioxygenase 1 (IDO1) is a novel tumor immunotherapy target, and its inhibitors show promising clinical applications, especially when used in combination with other immunostimulants. This article introduces two robust cell-based assays for screening IDO1 inhibitors. These assays are easy to use in most laboratories and can be used for screening IDO1 inhibitors. In cancer cell lines, endogenous IDO1 expression was induced using interferon-γ, and its activity was assessed by measuring kynurenine secreted into the culture medium. This study used the Jurkat T cell line to evaluate the effects of IDO1 induction and inhibition on T cell activation in cancer cells through co-culture experiments. Indicators for assessing cell viability were also used to detect false positives and toxic compounds of IDO1 inhibitors at an early stage. Clinical candidates epacadostat and BMS-986205 were used as control compounds in the experiments. epacadostat completely inhibited IDO1 activity, while BMS-986205 showed limited maximum inhibition (approximately 80% in this experimental system), consistent with their different interactions with IDO1. Both compounds at nanomolar concentrations reversed IDO1-mediated inhibition of T cell activation. However, treatment with micromolar concentrations of BMS-986205 blocked Jurkat T cell activation and induced cell death after prolonged incubation. [1]

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Background: IDO1 is highly expressed in a variety of cancers, and its metabolites can inhibit T cell function, potentially serving as a mechanism for tumor immune escape. Nivolumab (Nivo) is a monoclonal antibody targeting PD-1 that upregulates IDO1 expression, providing a theoretical basis for its combination with IDO1 inhibitors. Our preclinical study aimed to screen for the best IDO1 inhibitor with favorable pharmacokinetic (PK) characteristics (Hunt J et al., AACR 2017 [Abstract 6774]). This article introduces BMS-986205, a selective IDO1 inhibitor, validated in a new phase 1/2a clinical trial evaluating the efficacy of BMS-986205 as monotherapy and in combination with nivolumab. Methods: In the dose-escalation phase, patients with advanced cancer were grouped according to dose escalation, initially receiving BMS-986205 orally once daily (25-200 mg, as of January 5, 2017) for 2 weeks, followed by intravenous administration of BMS-986205 in combination with nivolumab 240 mg every 2 weeks. Primary study objectives included safety, pharmacokinetics, and pharmacodynamics. Preclinical analyses included enzyme inhibition assays in HEK293 cells overexpressing human IDO1 or tryptophan 2,3-dioxygenase (TDO) and IFNγ-stimulated HeLa cells. Results: Preclinical evaluations of BMS-986205 were performed to support the clinical trial. In vitro properties include: potent inhibition of kyn production in IDO1-HEK293 cells (IC50 = 1.1 nM), but no inhibitory effect on TDO-HEK293 cells; persistent inhibition in IDO1-based assays after elution; and single-digit nanomolar potency in human tolerable mixed lymphocyte response (MLR) assays. Based on preclinical data, a once-daily human dose of 150 mg is expected to maximally inhibit IDO1. In the ongoing clinical study, 42 patients have been treated. All treatment-related adverse events were grade 1/2, except for 3 cases of grade 3 toxicities (autoimmune hepatitis [dose-limiting; BMS-986205 100 mg/nivo 240 mg], rash, and asymptomatic hypophosphatemia). Starting with a 25 mg QD dose, individual trough concentrations began to exceed the whole blood IC50 value on day 14; starting with a 50 mg QD dose, trough concentrations began to exceed the IC90 value; and all patients receiving 200 mg QD treatment had trough concentrations exceeding the IC90 value. Serum kynurenine levels were significantly reduced in all dose groups (mean reduction >45% across all dose groups), with a mean reduction >60% in the 100 mg and 200 mg QD dose groups. Importantly, intratumoral kynurenine levels were reduced by up to 90% in evaluable paired pre-treatment and treatment-intervention samples. Conclusion: BMS-986205 is an optimized, once-daily, selective, and potent oral IDO1 inhibitor with good efficacy at clinically relevant concentrations. In this novel trial, BMS-986205, in combination with nivolumab, was well-tolerated at doses up to 200 mg. Even at a dose of 25 mg once daily, a significant reduction in serum kyn levels was observed; the inhibitory effects at doses of 100 mg and 200 mg once daily appeared to be superior to other similar compounds. Furthermore, we demonstrated for the first time that IDO1 inhibitors can reduce intratumoral kyn levels. These data suggest that BMS-986205 has excellent pharmacodynamic properties and is a promising IDO1 inhibitor, supporting further evaluation of its efficacy in combination with nivolumab. [2]

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Tumors can utilize the indoleamine 2,3-dioxygenase 1 (IDO1) pathway to construct an immunosuppressive microenvironment. Activated IDO1 metabolizes tryptophan to kyn, which has immunosuppressive effects, leading to suppression of effector T cell (Teff) proliferation, thereby allowing tumors to evade immune surveillance. IDO1 inhibitors can counteract this immunosuppressive tumor microenvironment and may improve cancer prognosis, especially when used in combination with other immunotherapies. Linrodostat mesylate is a potent, selective oral IDO1 inhibitor that blocks further IDO1 activation by occupying the heme cofactor binding site. It is currently undergoing multiple clinical trials for the treatment of advanced cancer patients. This article evaluates the in vitro potency, in vivo pharmacodynamic (PD) activity, and preclinical pharmacokinetics (PK) of linrodostat. Linrodostat exhibits potent cellular activity, inhibiting kynurenine production in HEK293 cells overexpressing human IDO1 and IFNγ-stimulated HeLa cells, without detecting any activity against tryptophan 2,3-dioxygenase or mouse indoleamine 2,3-dioxygenase 2. Linrodostat restores T cell proliferation in the mixed lymphocyte response to allogeneic dendritic cells expressing IDO1. In vivo experiments show that linrodostat reduces kynurenine levels in a human tumor xenograft model and exhibits significant pharmacodynamic activity. Linrodostat showed pharmacokinetic/pharmacodynamic relationships in xenograft models, preclinical animal models, and advanced cancer patient samples. It had high oral bioavailability in preclinical animal models and low to moderate systemic clearance. Our data suggest that linrodostat can effectively and specifically inhibit IDO1, thereby blocking an immunosuppressive mechanism that may be the reason why tumors evade host immune surveillance, and has good PK/PD properties, supporting clinical development. [3]

C24H24CLFN2O

410.9116

410.156

C, 70.15; H, 5.89; Cl, 8.63; F, 4.62; N, 6.82; O, 3.89

1923833-60-6

1923833-60-6;2221034-29-1 (mesylate);1791396-46-7 (dimesylate );

121328278

White to off-white solid powder

6.5

1

3

4

29

545

1

ClC1C=CC(=CC=1)NC([C@H](C)C1CCC(C2C=CN=C3C=CC(=CC=23)F)CC1)=O

KRTIYQIPSAGSBP-KLAILNCOSA-N

InChI=1S/C24H24ClFN2O/c1-15(24(29)28-20-9-6-18(25)7-10-20)16-2-4-17(5-3-16)21-12-13-27-23-11-8-19(26)14-22(21)23/h6-17H,2-5H2,1H3,(H,28,29)/t15-,16?,17?/m1/s1

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: 2.5 mg/mL (6.08 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (6.08 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.06 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 20.8 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.

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Solubility in Formulation 4: 2.08 mg/mL (5.06 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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.

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Solubility in Formulation 5: ≥ 2.08 mg/mL (5.06 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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