JAK3 (Ki = 2.5 nM); JAK1 (Ki = 11 nM); Tyk2 (Ki = 11 nM); JAK2 (Ki = 13 nM); FLT3 (Ki = 1 μM); ROCK I (Ki = 1.5 μM); GSK3β (Ki = 1.8 μM); CDK2/CycA (Ki = 2.6 μM); PknB (Ki = 8 μM)

Strong JAK3 inhibitor decernotinib (VX-509) has Ki values of 2.5, 11, 13, and 11 nM for JAK3, JAK1, JAK2, and TYK2, in that order. With a mean IC50 of 170 ± 101 nM, decernotinib potently inhibits T cell proliferation. It also inhibits T cell proliferation driven by IL-2 (IC50, 140, and 400 nM). In reaction to CD40L and IL-4, VX-509 is also cytotoxic (IC50, 50 nM) against B cells [1].

In rats injected with collagen, decernotinib (VX-509, 10, 25, or 50 mg/kg, po) substantially and dose-dependently reduced the increases in ankle diameter and paw weight. In rats, decernotinib effectively lowers bone resorption and cartilage degradation. Decernotinib (10, 25, or 50 mg/kg, po, bid) reduces delayed-type hypersensitivity-related ear edema in a mouse model [1].

The effect of Decernotinib (VX-509; VRT-831509) on JAK3 activity was assessed by measuring the residual kinase activity of the recombinantly expressed JAK3 kinase domain using a radiometric assay. The final concentrations of the components in the assay wer as follows: 100 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol (DTT), 0.01% BSA, 0.25 nM JAK3, 0.25 mg/mL polyE4Y, and 5 μM 33P-γ-ATP (200 µCi/µmol). A 10 mM stock solution of Decernotinib (VX-509; VRT-831509) is prepared in DMSO, from which additional dilutions are prepared. A substrate mixture (100 mM HEPES, 10 mM MgCl2, 0.5 mg/mL polyE4Y, and 10 μM 33P-γ-ATP) is added and mixed with Decernotinib (VX-509; VRT-831509) stock solution. The reaction is initiated by the addition of an enzyme mixture [100 mM HEPES (pH 7.5), 10 mM MgCl2, 2 mM DTT, 0.02% BSA, 0.5 nM JAK3]. After 15 minutes, the reaction is quenched with 20% trichloroacetic acid (TCA). The quenched reaction is transferred to the GF/B filter plates and washed three times with 5% TCA. Following the addition of Ultimate Gold scintillant (50 μL), the samples are counted in a Packard TopCount gamma counter. In this procedure, the radioactivity trapped is a measure of the residual JAK3 kinase activity. From the activity versus concentration of Decernotinib (VX-509; VRT-831509)titration curve, the Ki value is determined by fitting the data to an equation for competitive tight binding inhibition kinetics[1].

Whole-blood samples from healthy volunteers were used to collect peripheral blood mononuclear cells, which are plated in T75 tissue culture flasks at a density of 1 × 106/mL. Cells are stimulated with 10 μg/mL phytohemagglutinin at 37°C for 72 hours. After 72 hours, cells wer detached from the flask by scraping, washed, and plated at a density of 1 × 105/well in a 96-well plate. Decernotinib (VX-509; VRT-831509) (9.7 nM to 10 μM) is added, and plates were incubated for 30 minutes at 37°C, followed by stimulation with human IL-2. In two rows, only DMSO is added; one row is not stimulated with IL-2, and one row is stimulated with IL-2 to serve as the proliferation control. Plates are incubated at 37°C for 2 days. On day 2, cells were pulsed with 20 µCi/mL methyl-[3H]thymidine for 18-24 hours and harvested onto filters for radiographic determination. Data are analyzed to generate an IC50 value using Softmax pro software[1]

The collagen-induced arthritis (CIA) rat model was used to evaluate the effects of oral Decernotinib (VX-509; VRT-831509; adelatinib) [10 mg/kg b.i.d., 25 mg/kg b.i.d., 50 mg/kg b.i.d., 50 mg/kg q.d., or 100 mg/kg q.d.] on joint inflammation and histopathology. Female Lewis rats (157-187 g) are anesthetized with isoflurane and injected with 300 µL Freund’s incomplete adjuvant, containing 2 mg/mL bovine type II collagen, at the base of the tail and two sites on the back on days 0 and 6. The rats are randomized to study groups at the onset of paw swelling (arthritis), which occurs between days 10 and 11. Dosing of either Decernotinib (VX-509; VRT-831509; adelatinib) or vehicle via oral gavage is initiated on the first day of established arthritis and continued to day 6 of arthritis. Dosing volume is 5 mL/kg. Groups are controls (no collagen injection plus vehicle; n = 4), collagen plus vehicle (n = 5), collagen plus Decernotinib (VX-509; VRT-831509; adelatinib) 10 mg/kg b.i.d. (n = 8); collagen plus Decernotinib (VX-509; VRT-831509; adelatinib) 10 mg/kg b.i.d. (n = 8); collagen plus (n = 8); collagen plus (n = 8); collagen plus (n = 8); collagen plu (n = 8); and collagen plus (n = 8); collagen plu (n = 8); all treatments are administered for 6 days. An additional group of rats is given collagen plus 10 mg/kg subcutaneous etanercept, a human tumor necrosis factor-α antagonist, on study days 11 and 14. Caliper measurements of normal (baseline) ankle joints begin on day 9 and continue through the last day of study. Differences in mean ankle diameter are tested for significance using Student’s t test, with significance set at P ≤ 0.05. The rats are euthanized on day 7 of arthritis, which is study day 17 or 18 depending on when animals are randomized to groups; paws and knees are harvested to determine paw weight and to conduct a histopathological analysis of inflammation (knee and ankle), pannus formation (ankle), cartilage destruction (knee), and bone resorption (knee and ankle). Scores range from 0 (normal) to 5 (severe pathology) and are assigned by a veterinary pathologist. Percent inhibition is calculated using the following formula: [(mean of treatment group) − (mean of control)] ÷ [(mean of collagen + vehicle) − (mean of control)]. Kruskal-Wallis one-way analysis of variance nonparametric tests are used to determine statistical significance among the histopathology groups, with significance set at P ≤ 0.05[1].

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Dissolved in 10% vitamin E d-α-tocophenyl polyethylene glycol 1000 succinate and 1% hydroxypropyl methylcellulose acetyl succinate; 50 mg/kg; administrated orally

Collagen-induced arthritis (CIA) rat model

[1]. VX-509 is a potent and selective Janus kinase 3 (JAK3) inhibitor that attenuates inflammation in animal models of autoimmune disease. J Pharmacol Exp Ther. 2015 Mar 11. pii: jpet.114.221176.

Decernotinib has been used in trials studying the treatment of Drug Interactions and Rheumatoid Arthritis.
DECERNOTINIB is a small molecule drug with a maximum clinical trial phase of II and has 1 investigational indication.

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Cytokines, growth factors, and other chemical messengers rely on a class of intracellular nonreceptor tyrosine kinases known as Janus kinases (JAKs) to rapidly transduce intracellular signals. A number of these cytokines are critical for lymphocyte development and mediating immune responses. JAK3 is of particular interest due to its importance in immune function and its expression, which is largely confined to lymphocytes, thus limiting the potential impact of JAK3 inhibition on nonimmune physiology. The aim of this study was to evaluate the potency and selectivity of the investigational JAK3 inhibitor VX-509 (decernotinib) [(R)-2-((2-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)-2-methyl-N-(2,2,2-trifluoroethyl)butanamide] against JAK3 kinase activity and inhibition of JAK3-mediated signaling in vitro and JAK3-dependent physiologic processes in vivo. These results demonstrate that VX-509 potently inhibits JAK3 in enzyme assays (Ki = 2.5 nM + 0.7 nM) and cellular assays dependent on JAK3 activity (IC50 range, 50-170 nM), with limited or no measurable potency against other JAK isotypes or non-JAK kinases. VX-509 also showed activity in two animal models of aberrant immune function. VX-509 treatment resulted in dose-dependent reduction in ankle swelling and paw weight and improved paw histopathology scores in the rat collagen-induced arthritis model. In a mouse model of oxazolone-induced delayed-type hypersensitivity, VX-509 reduced the T cell-mediated inflammatory response in skin. These findings demonstrate that VX-509 is a selective and potent inhibitor of JAK3 in vitro and modulates proinflammatory response in models of immune-mediated diseases, such as collagen-induced arthritis and delayed-type hypersensitivity. The data support evaluation of VX-509 for treatment of patients with autoimmune and inflammatory diseases such as rheumatoid arthritis.[1]

C18H19F3N6O

392.38

392.157

C, 55.10; H, 4.88; F, 14.53; N, 21.42; O, 4.08

944842-54-0

59422203

White to off-white solid powder

1.4±0.1 g/cm3

553.6±50.0 °C at 760 mmHg

288.6±30.1 °C

0.0±1.5 mmHg at 25°C

1.603

2.26

3

8

6

28

548

1

CC[C@](C)(C(=O)NCC(F)(F)F)NC1=NC(=NC=C1)C2=CNC3=C2C=CC=N3

ASUGUQWIHMTFJL-QGZVFWFLSA-N

InChI=1S/C18H19F3N6O/c1-3-17(2,16(28)25-10-18(19,20)21)27-13-6-8-23-15(26-13)12-9-24-14-11(12)5-4-7-22-14/h4-9H,3,10H2,1-2H3,(H,22,24)(H,25,28)(H,23,26,27)/t17-/m1/s1

(R)-2-((2-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)-2-methyl-N-(2,2,2-trifluoroethyl)butanamide

PubChem CID 59422203; VX-509; VRT831509 ; 944842-54-0; Adelatinib; Decernotinib (VX-509); Decernotinib(VX-509); VRT-831509; VX509; VX 509 ; VRT 831509 ; Decernotinib; Adelatinib

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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 (6.37 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 25.0 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 2: ≥ 2.5 mg/mL (6.37 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 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 3: ≥ 2.5 mg/mL (6.37 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 25.0 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.)