PF-06840003

Catalog No.S8657 Batch:S865701

Technical Data

Formula

C₁₂H₉FN₂O₂

Molecular Weight

232.21

CAS No.

198474-05-4

Solubility (25°C)*

In vitro

DMSO

46 mg/mL (198.09 mM)

Ethanol

6 mg/mL (25.83 mM)

Water

Insoluble

In vivo (Add solvents to the product individually and in order)

Clear solution	5% DMSO 1 95% corn oil	0.6mg/ml	Taking the 1 mL working solution as an example, add 50 μL of 12 mg/ml clear DMSO stock solution to 950 μL of corn oil and mix evenly. The mixed solution should be used immediately for optimal results.
Clear solution	5%DMSO 1 40%PEG300 2 5%Tween80 3 50%ddH2O	20.0mg/ml	Taking the 1 mL working solution as an example, add 50 μL of 400 mg/ml clarified DMSO stock solution to 400 μL PEG300, mix evenly to clarify it; add 50 μL Tween80 to the above system, mix evenly to clarify it; then continue to add 500 μL ddH2O to adjust the volume to 1 mL. The mixed solution should be used immediately for optimal results.

* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
* Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

Preparing Stock Solutions

Biological Activity

Description

PF-06840003 (EOS200271) is a highly selective orally bioavailable IDO-1 inhibitor. Although it has moderate hIDO1 enzyme inhibition (IC50 0.41 μM), it is a highly efficient compound (LE 0.53, LipE 5.1), driven by its tight packing within the enzyme, as well as the high density of hydrogen bonds it forms with hIDO-1 despite its small size.

Targets

hIDO-1 ^[1] (Cell-free)	dIDO-1 ^[1] (Cell-free)	mIDO-1 ^[1] (Cell-free)
0.41 μM	0.59 μM	1.5 μM

In vitro

PF-06840003 is a racemic mixture. The IC50s of PF-06840003 for hIDO-1, mouse IDO-1 and dog IDO-1 are 0.41, 1.5 and 0.59 μM, respectively. It has very weak activity against hTDO-2, with an IC50 of 140 μM. In cellular assays, PF-06840003 shows activity both in the HeLa assay (IC50 1.8 μM) as well as in the LPS/INFγ-stimulated THP1 cells (IC50 1.7 μM). PF-06840003 is a very weak inhibitor of CYPs with IC50 values greater than 100 μM for most major CYP isozymes except 2C19 (IC50 value is 78 μM). Additionally, PF-06840003 does not exhibit metabolism-dependent (time-dependent and NADPH-dependent or time-dependent (NADPH-independent) inhibition of the major CYP enzymes investigated^[1].

In vivo

PF-06840003 has a predicted half-life of 16−19 h. Oral bioavailability in the mouse and rat was 59 and 94%, respectively, and 19% in dog. PF-06840003 has shown significant antitumor activity in monotherapy in Pan02, B16−F10, CT26, MC38, 4T1, and Renca models (p < 0.05 vs vehicle-treated group) and very good synergy in combination with anti-PDL1 mAb in CT26 model (p < 0.05 vs monotherapy groups).The PK profile of the compound is excellent, with a low/moderate clearance in most preclinical species. The compound also shows good CNS penetration in rat, suggesting potential impact on brain metastases^[1].

Protocol (from reference)

Cell Assay:^{^[1]}	Cell lines HeLa cervical carcinoma cells Concentrations 0-50 μM Incubation Time 16-24 h Method HeLa cells were harvested from cell culture flasks using 0.25% trypsin/EDTA and neutralized with EMEM growth medium. Following resuspension in fresh growth media, cells were seeded at 20,000 cells per well in 200 μL growth media in a 96-well plate and allowed to adhere at 37 °C at 5% CO2 overnight. The following day, growth media was aspirated and replaced with 200 μL of reduced (2%) serum media containing 100 ng/mL recombinant human interferon gamma (rhIFNγ) and incubated at 37 °C with 5% CO2 for 48 h to induce IDO-1 expression. On day four, compounds were diluted to 10 mM in DMSO and 11-point three-fold dilutions were prepared. rhIFNγ-containing media was removed, and following dilution into EMEM, compounds were added to cells at 50 μM top concentration and allowed to incubate 16−24 h at 37 °C with 5% CO2. On day five of the assay, 100 μL of cell supernatant was transferred to a v-bottom 96-well plate. Thirty microliters of 30% trichloroacetic acid (TCA) was added to each well to precipitate proteins, and plates were centrifuged at 3000 rpm for 10 min. One hundred microliters was transferred to a fresh flat-bottom 96-well plate, and 100 μL/well of 2% 4-(dimethylamino)benzaldehyde (pDMAB) in acetic acid was added to derivatize N-formyl kynurenine to kynurenine for quantitative colorimetric readout. Assay plates were read at A492 on Envision plate reader.
Animal Study:^{^[1]}	Animal Models CD-1 mice, Wistar−Han rat, and Beagle dog Dosages 1 mg/kg (IV) and 3 mg/kg (oral) Administration i.v./oral

Cell Assay:^{^[1]}

Cell lines
HeLa cervical carcinoma cells
Concentrations
0-50 μM
Incubation Time
16-24 h
Method
HeLa cells were harvested from cell culture flasks using 0.25% trypsin/EDTA and neutralized with EMEM growth medium. Following resuspension in fresh growth media, cells were seeded at 20,000 cells per well in 200 μL growth media in a 96-well plate and allowed to adhere at 37 °C at 5% CO2 overnight. The following day, growth media was aspirated and replaced with 200 μL of reduced (2%) serum media containing 100 ng/mL recombinant human interferon gamma (rhIFNγ) and incubated at 37 °C with 5% CO2 for 48 h to induce IDO-1 expression. On day four, compounds were diluted to 10 mM in DMSO and 11-point three-fold dilutions were prepared. rhIFNγ-containing media was removed, and following dilution into EMEM, compounds were added to cells at 50 μM top concentration and allowed to incubate 16−24 h at 37 °C with 5% CO2. On day five of the assay, 100 μL of cell supernatant was transferred to a v-bottom 96-well plate. Thirty microliters of 30% trichloroacetic acid (TCA) was added to each well to precipitate proteins, and plates were centrifuged at 3000 rpm for 10 min. One hundred microliters was transferred to a fresh flat-bottom 96-well plate, and 100 μL/well of 2% 4-(dimethylamino)benzaldehyde (pDMAB) in acetic acid was added to derivatize N-formyl kynurenine to kynurenine for quantitative colorimetric readout. Assay plates were read at A492 on Envision plate reader.

Animal Study:^{^[1]}

Animal Models
CD-1 mice, Wistar−Han rat, and Beagle dog
Dosages
1 mg/kg (IV) and 3 mg/kg (oral)
Administration
i.v./oral

References

[1] Crosignani S, et al. J Med Chem. 2017, 60(23):9617-9629.

Selleck's PF-06840003 has been cited by 5 publications

25-hydroxyvitamin D3 generates immunomodulatory plasticity in human periodontal ligament-derived mesenchymal stromal cells that is inflammatory context-dependent [ Front Immunol, 2023, 14:1100041]	PubMed: 36761739
System analysis based on the cancer-immunity cycle identifies ZNF207 as a novel immunotherapy target for hepatocellular carcinoma [ J Immunother Cancer, 2022, 10(3)e004414]	PubMed: 35246476
IL4I1 Is a Metabolic Immune Checkpoint that Activates the AHR and Promotes Tumor Progression [ Cell, 2020, 182(5):1252-1270.e34]	PubMed: 32818467
Pleiotropic effects of vitamin D3 on CD4+ T lymphocytes mediated by human periodontal ligament cells and inflammatory environment. [ J Clin Periodontol, 2020, 10.1111/jcpe.13283]	PubMed: 32160330
Cytokines Differently Define the Immunomodulation of Mesenchymal Stem Cells From the Periodontal Ligament [ Cells, 2020, 14;9(5):E1222]	PubMed: 32423044

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