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.

Linrodostat PK profile in vivo [3]
To extend these SKOV3 xenograft studies, we assessed PK parameters of IV and oral linrodostat in higher species including rat, dog, and monkey (Table 1). After IV administration, the CLTp of linrodostat was comparable across species, with levels of 27, 25, and 19 mL/min/kg in rats, dogs, and monkeys, respectively. The CLTp were ≤ 48% of the respective reported liver blood flows, indicating that linrodostat has low-to-moderate systemic clearance. After IV administration, Vss values in rats, dogs, and monkeys were 3.8, 5.7, and 4.1 L/kg, respectively, indicating extravascular distribution. The t1/2 of linrodostat was 3.9, 4.7, and 6.6 hours in rats, dogs, and monkeys, respectively. After oral administration, the Tmax ranged from 0.5 to 1.7 hours in rats, dogs, and monkeys. The absolute oral bioavailability of linrodostat given as a solution was 64%, 39%, and 10% in rats, dogs, and monkeys, respectively. The rate of linrodostat metabolism by hepatocytes was higher in rats than dogs, monkeys, and humans. Based on the in vitro–in vivo correlation of hepatocyte clearance in animal species, clearance in humans is projected to be moderate. These data lead to a projected human t1/2 of 23 hours, supporting dosing once a day in a clinical setting.
To extend these preclinical, in vivo PD/PK studies, we assessed kynurenine PD with linrodostat + nivolumab treatment in patients with advanced cancers. Substantial decreases in mean serum kynurenine levels were detected at all linrodostat doses evaluated, with an approximately 60% reduction observed with 100 and 200 mg once a day (Fig. 5A and B). Furthermore, pre- and on-treatment tumor samples from 13 patients demonstrated that intratumoral kynurenine levels were reduced across all doses following treatment, even in the presence of relatively high pretreatment kynurenine levels (Fig. 5C). These results demonstrate that linrodostat + nivolumab leads to substantial reductions in 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 under investigation in clinical trial NCT03247283 (Pharmacokinetics and Metabolism Study in Healthy Male Participants).
Linrodostat is an orally available inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1), with potential immunomodulating and antineoplastic activities. Upon administration, linrodostat specifically targets and binds to IDO1, a cytosolic enzyme responsible for the oxidation of the amino acid tryptophan into the immunosuppressive metabolite kynurenine. By inhibiting IDO1 and decreasing kynurenine 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 causes a reduction in tumor-associated regulatory T-cells (Tregs). Activation of the immune system, which is suppressed in many cancers, may induce a cytotoxic T-lymphocyte (CTL) response against the IDO1-expressing tumor cells, thereby inhibiting the growth of IDO1-expressing tumor cells. IDO1, overexpressed by multiple tumor cell types, plays an important role in immunosuppression. Tryptophan depletion inhibits T-lymphocyte proliferation and activation, and subsequently suppresses the immune system.

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Indoleamine 2,3-dioxygenase 1 (IDO1) is a new immune-oncology target and its inhibitors have shown promise in the clinic especially in combination with other immune-stimulating agents. Here we describe two robust cell-based assays for screening IDO1 inhibitors. Both assays can be easily adopted by most laboratories and utilized for screening of IDO1 inhibitors. Endogenous IDO1 expression is induced in a cancer cell line with interferon gamma and its activity is assessed by measuring kynurenine secreted into the media. The effect of cancer cell IDO1 induction and inhibition on T cell activation is evaluated in a co-culture assay using Jurkat T cell line. Additional readouts assessing cell viability are employed for early detection of false positive IDO1 inhibitors and toxic compounds. Clinical candidates epacadostat and BMS-986205 were evaluated in the assays as control compounds, the former can completely inhibit IDO1 activity while the maximum effect of the later is limited (to about 80% in our system) consistent with the differences in their interaction with IDO1. Nanomolar concentrations of both compounds rescued IDO1 mediated inhibition of T cell activation. However, treatment with micromolar concentrations of BMS-986205 blocked Jurkat T cell activation and after prolonged incubation induced cell death.[1]

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Background: IDO1 is highly expressed in multiple cancers and may be an immunosuppressive mechanism for tumor escape via its production of metabolites that inhibit T-cell function. Nivo, a mAb that targets PD-1, causes IDO1 upregulation, supporting a rationale for combining it with an IDO1 inhibitor. Our preclinical program aimed to identify a best in class IDO1 inhibitor with favorable pharmacokinetic (PK) characteristics (Hunt J, et al. AACR 2017 [abst 6774]). Here we present BMS-986205, a selective IDO1 inhibitor validated in a novel phase 1/2a trial alone and in combination with nivo. Methods: 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. Preclinical analyses included measurement of enzyme inhibition in HEK293 cells overexpressing human IDO1 or tryptophan 2,3-dioxygenase (TDO) and IFNγ-stimulated HeLa cells. Results: 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. 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. Conclusions: BMS-986205 is an optimized, once-daily, selective and potent oral IDO1 inhibitor at clinically relevant concentrations. It is well tolerated up to at least 200 mg in combination with nivo in this novel trial. Evidence of substantial serum kyn reduction was observed at doses as low as 25 mg QD; inhibition at 100 and 200 mg QD appears greater than that reported for other in-class compounds. In addition, we have presented the first evidence of intratumoral kyn reduction by an IDO1 inhibitor. These data suggest the potential of BMS-986205 as an IDO1 inhibitor with superior PD properties and support further evaluation in combination with nivo.[2]

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Tumors can exploit the indoleamine 2,3-dioxygenase 1 (IDO1) pathway to create an immunosuppressive microenvironment. Activated IDO1 metabolizes tryptophan into immunosuppressive kynurenine, leading to suppressed effector T-cell (Teff) proliferation, allowing for tumor escape from host immune surveillance. IDO1 inhibition counteracts this immunosuppressive tumor microenvironment and may improve cancer outcomes, particularly when combined with other immunotherapies. Linrodostat mesylate (linrodostat) is a potent, selective oral IDO1 inhibitor that occupies the heme cofactor-binding site to prevent further IDO1 activation and is currently in multiple clinical trials for treatment of patients with advanced cancers. Here, we assess the in vitro potency, in vivo pharmacodynamic (PD) activity, and preclinical pharmacokinetics (PKs) of linrodostat. 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. In vivo, linrodostat reduced kynurenine levels in human tumor xenograft models, exhibiting significant PD activity. Linrodostat demonstrated a PK/PD relationship in the xenograft model, preclinical species, and samples from patients with advanced cancers, with high oral bioavailability in preclinical species and low to moderate systemic clearance. Our data demonstrate that linrodostat potently and specifically inhibits IDO1 to block an immunosuppressive mechanism that could be responsible for tumor escape from host immune surveillance with favorable PK/PD characteristics that support 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.)