Delgocitin potently inhibits all JAK subtypes in the enzymatic assays; the IC50 values for JAK1, JAK2, JAK3, and Tyk2 are 2.8±0.6, 2.6±0.2, 13±0, and 58±9 nM, respectively. Lineweaver-Burk plots demonstrate that dexgocitinib inhibits all JAKs in a competitive manner with ATP, with Ki values for JAK1, JAK2, JAK3, and Tyk2 being 2.1±0.3, 1.7±0.0, 5.5±0.3, and 14±1 nM, respectively. Delgocitinib had IC50 values of 40±9, 33±14, 84±11, 304±22, and 18±3 nM, respectively, to block the phosphorylation of Stat proteins produced by IL-2, IL-6, IL-23, GM-CSF, and IFN-α in these cell-based cytokine signaling tests. Similar in potency to CP-690550 (IC50=16 nM), delogocitinib likewise inhibits T cell proliferation caused by IL-2 in a concentration-dependent manner (IC50=8.9±3.6 nM)[1].

IFN-γ production is decreased by delognocitinib; however, the potency of the treatment one hour previously is greater than that of the administration six hours prior (ED50=0.24 versus 1.3 mg/kg). Delgocitinib, when taken as prescribed from day 1, inhibits the growth of edema in the hind paw as well as histological alterations such as synovial cell hyperplasia and inflammatory cell infiltration. Delgocitinib prevents bone and cartilage deterioration seen in radiography and histology. When administered starting on day 15, delocitinib reduces paw swelling in a way that is dose-dependent. Moreover, Delgocitinib reduces the degradation of cartilage and bone, hyperplasia of synovial cells, and infiltration of inflammatory cells in the histological and radiographic examinations at the conclusion of the study[1].

Enzyme assay[1]
Kinase assays using a recombinant enzyme, biotinylated substrate, and [33P]ATP were performed as previously described with some modifications. Each enzyme reaction was performed with a recombinant JAK1, JAK2, JAK3, and Tyk2 enzyme (4, 0.2, 0.2, and 2 μg/mL protein, respectively), 10 μM TK substrate-biotin, ATP at the Km specific for each enzyme (40, 10, 2.5, and 40 μM, respectively), 50 mM HEPES (pH 7.0), 0.01 % bovine serum albumin (BSA), 0.02 % NaN3, 0.1 mM orthovanadate, 5 mM MgCl2, 1 mM dithiothreitol, 50 nM Supplement Enzymatic Buffer, and 1 % DMSO.

Flow cytometry analysis[1]
hPBMCs (3 × 106 cells/tube) were incubated with a test compound for 30 min at 37 °C, and then treated with IL-2 (100 ng/mL), IL-6 (100 ng/mL), IL-23 (100 ng/mL), IFN-α (100 ng/mL), or GM-CSF (1 ng/mL) for an additional 15 min. To terminate the stimulation, the cells were fixed with Fixation Buffer for 10 min at 37 °C. The fixed cells were incubated with Perm Buffer II for 30 min on ice and then incubated with fluorochrome-labeled anti-CD3, anti-CD4, or anti-phospho-Stat antibodies for 30 min at room temperature. The cytokine-induced Stat phosphorylation was analyzed and quantified using a Cytomics FC500. In the case of IL-23 stimulation, hPBMCs were precultured with 10 μg/mL phytohemagglutinin (PHA)-M for 3 days to enhance their response to IL-23.

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Cellular assays[1]
For determination of IL-2-induced T cell proliferation, human T cells were precultured with 10 μg/mL PHA-M for 3 days and plated in 96-well plates at 1.0 × 104 cells/well in the presence or absence of various concentrations of JTE-052. Following preincubation with the compound for 30 min at 37 °C, the cells were stimulated by adding 20 ng/mL recombinant human IL-2 to each well and incubated for 3 days at 37 °C under 5 % CO2. [3H]Thymidine (37 kBq) was added during the culture period. After completion of the culture period, the cells were harvested with a 96-well harvester and counted in a scintillation counter.

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For determination of IL-21-induced B cell proliferation, human B cells were plated in 96-well plates at 1 × 105 cells/well in the presence or absence of JTE-052. Following preincubation with the compound for 30 min, the cells were stimulated by adding 10 ng/mL recombinant human IL-21 and 1 μg/mL anti-CD40 antibody. The cells were then cultured for 3 days, and [3H]thymidine uptake was measured as described above. For determination of GM-CSF-induced tumor necrosis factor (TNF)-α production from monocytes, human monocytes were plated in 96-well plates at 1 × 105 cells/well in the presence or absence of JTE-052. Following preincubation with the compound for 30 min, the cells were stimulated by adding 0.03 ng/mL recombinant human GM-CSF for 16 h. The cells were then cultured with 0.1 ng/mL lipopolysaccharide (LPS) for an additional 6 h at 37 °C. The supernatants were collected, and secretion of TNF-α was measured by ELISA. For determination of IL-4-induced TNF-α production from mast cells, human mast cells were plated in 96-well plates at 2 × 105 cells/well in the presence or absence of JTE-052. Following preincubation with the compound for 10 min, the cells were stimulated by adding 10 ng/mL recombinant human IL-4 and 1 μg/mL immunoglobulin (Ig)E for 5 days. The cells were then replated at 1.2 × 105 cells/well and cultured with 1 μg/mL anti-IgE for an additional 5 h at 37 °C. The supernatants were collected, and secretion of TNF-α was measured by ELISA. For determination of cytotoxicity, fibroblast proliferation was measured. NHLFs were plated in 96-well plates at 1 × 103 cells/well in the presence or absence of JTE-052 without the addition of cytokines. The cells were then cultured for 3 days, and [3H]thymidine uptake was measured as described above. The cells were cultured at 37 °C under 5 % CO2 in RPMI1640 medium containing 10 % fetal calf serum (T cells, B cells, monocytes, and NHLFs) or Iscove’s modified Dulbecco’s medium containing 0.1 % BSA, 55 µM 2-mercaptoethanol, and 1 % Insulin-Transferrin-Selenium, 100x (Mast cells).

IL-2-induced IFN-γ production in mice[1]
DBA/1J mice were dosed once via oral gavage with compounds. At 1 or 6 h after compound administration, the mice were injected intraperitoneally with 2 μg of recombinant mouse IL-2 and 10 μg of biotinylated anti-mouse IFN-γ capture antibody. After 6 h, the mice were euthanized, and blood was collected. Serum IFN-γ was quantified using a Mouse IFN-γ In Vivo Capture Assay Set.

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Collagen-induced arthritis in rats[1]
CIA was induced in rats as previously described with some modifications. Briefly, type II collagen was dissolved in 0.01 M acetic acid at 2 mg/mL, and the solution was emulsified in an equal volume of Freund’s incomplete adjuvant. Lewis rats were immunized with 1 mL of the emulsion (1 mg of type II collagen) via ten intradermal injections on the back under anesthesia. The rats were then challenged with 0.2 mL of the emulsion injected into the base of the tail on day 8 under anesthesia. The test drugs were given orally once daily from day 1 to day 21 (preventive administration) or from day 15 to day 28 (therapeutic administration). After arthritis induction, the hind paw volume was measured by a water displacement method using a plethysmometer. On day 22 or day 29, the rats were euthanized, and their hind paws were excised and X-rayed or processed for histological evaluation. Radiographs of the right hindlimbs were taken using a microfocal cone-beam X-ray CT scanner. The severity of bone destruction was scored on a 3-point scale ranging from 0 to 2 for the following five joints: second metatarsal—second intertarsal joint, third metatarsal—third intertarsal joint, talus—central intertarsal joint, calcaneus—fourth intertarsal joint, and astragalo—tibial joint. For histological analysis, the formalin-fixed left hindlimbs were sectioned and stained with hematoxylin and eosin. The histology of the tarsal joints was evaluated by assessing the following parameters defined in the preliminary examination: inflammatory cell infiltration, synovial cell hyperplasia, cartilage destruction, and bone destruction. The severity of each histological change was scored on a 5-point scale ranging from 0 to 4. (Score 0: normal; Score 1: minimal, solitary (and very small) lesion; Score 2: slight, focal (and small) lesion; Score 3: moderate, scattered lesion; and Score 4: severe, extensive lesion).

[1]. Tanimoto A, et al. Pharmacological properties of JTE-052: a novel potent JAK inhibitor that suppresses various inflammatory responses in vitro and in vivo. Inflamm Res. 2015 Jan;64(1):41-51

Objective: To evaluate the pharmacological properties of JTE-052, a novel Janus kinase (JAK) inhibitor.[1]
Methods: The JAK inhibitory activity of JTE-052 was evaluated using recombinant human enzymes. The inhibitory effects on cytokine signaling pathways were evaluated using primary human inflammatory cells. The in vivo efficacy and potency of JTE-052 were examined in a mouse interleukin (IL)-2-induced interferon (IFN)-γ production model and a rat collagen-induced arthritis model.
Results: JTE-052 inhibited the JAK1, JAK2, JAK3, and tyrosine kinase (Tyk)2 enzymes in an adenosine triphosphate (ATP)-competitive manner and inhibited cytokine signaling evoked by IL-2, IL-6, IL-23, granulocyte/macrophage colony-stimulating factor, and IFN-α. JTE-052 inhibited the activation of inflammatory cells, such as T cells, B cells, monocytes, and mast cells, in vitro. Oral dosing of JTE-052 resulted in potent suppression of the IL-2-induced IFN-γ production in mice with an ED50 value of 0.24 mg/kg, which was more potent than that of tofacitinib (ED50 = 1.1 mg/kg). In the collagen-induced arthritis model, JTE-052 ameliorated articular inflammation and joint destruction even in therapeutic treatments where methotrexate was ineffective.
Conclusions: The present results indicate that JTE-052 is a highly potent JAK inhibitor, and represents a candidate anti-inflammatory agent for suppressing various types of inflammation.

C16H18N6O

310.35372209549

310.15

C, 61.92; H, 5.85; N, 27.08; O, 5.15

1263774-59-9

2064338-33-4 (EtOH);1263774-59-9 (free);2064338-35-6 (CHCl3);

50914062

White to off-white solid powder

1.1

88.9Ų

N#CCC(N1C[C@H](C)[C@]12CN(C3=C4C(NC=C4)=NC=N3)CC2)=O

LOWWYYZBZNSPDT-ZBEGNZNMSA-N

InChI=1S/C16H18N6O/c1-11-8-22(13(23)2-5-17)16(11)4-7-21(9-16)15-12-3-6-18-14(12)19-10-20-15/h3,6,10-11H,2,4,7-9H2,1H3,(H,18,19,20)/t11-,16-/m0/s1

3-[(3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl]-3-oxopropanenitrile

LEO-124249 JTE-052 LEO 124249JTE 052LEO124249 JTE052

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 : ≥ 58 mg/mL (~186.89 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.06 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (8.06 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 0.5 mg/mL (1.61 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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