JAK2 (IC50 = 2.8 nM); JAK1 (IC50 = 3.3 nM); Tyk2 (IC50 = 19 nM); JAK3 (IC50 = 428 nM)

Ruxolitinib produces a dose-dependent increase in apoptosis, a doubling of cells with depolarized mitochondria in Ba/F3 cells, and a powerful and specific inhibition of JAK2V617F-mediated signaling and proliferation. Ruxolitinib reduced the proliferation of erythroid progenitor cells from normal donors and polycythemia vera patients with IC50 values of 407 nM and 223 nM, respectively, and demonstrated substantial anti-erythroid colony formation with an IC50 of 67 nM [1].

In JAK2V617F-driven mouse models, rufolitinib (180 mg/kg, PO, twice daily) did not cause myelosuppression or immunosuppression, but it did significantly prolong survival by reducing splenomegaly and circulating levels of inflammatory cytokines and preferentially eliminating tumor cells. At day 22, survival rates were above 90% [1]. In the myelofibrosis double-blind trial, 41.9% of patients in the ruxolitinib group and 0.7% of patients in the placebo group achieved the primary endpoint. Ruxolitinib improves overall symptom scores by 50% or more while maintaining spleen volume reduction [2].

Biochemical assays[1]
The kinase domains of human JAK1 (837-1142), JAK2 (828-1132), JAK3 (781-1124), and Tyk2 (873-1187) were cloned by PCR with N-terminal epitope tags. Recombinant proteins were expressed using Sf21 cells and baculovirus vectors and purified with affinity chromatography. JAK kinase assays used a homogeneous time-resolved fluorescence assay with the peptide substrate (-EQEDEPEGDYFEWLE). Each enzyme reaction was carried out with test compound or control, JAK enzyme, 500nM peptide, adenosine triphosphate (ATP; 1mM), and 2.0% dimethyl sulfoxide (DMSO) for 1 hour. The 50% inhibitory concentration (IC50) was calculated as the compound concentration required for inhibition of 50% of the fluorescent signal. Biochemical assays for CHK2 and c-MET enzymes were performed using standard conditions (Michaelis constant [Km] ATP) with recombinantly expressed catalytic domains from each protein and synthetic peptide substrates.
An additional panel of kinase assays (Abl, Akt1, AurA, AurB, CDC2, CDK2, CDK4, CHK2, c-kit, c-Met, EGFR, EphB4, ERK1, ERK2, FLT-1, HER2, IGF1R, IKKα, IKKβ, JAK2, JAK3, JNK1, Lck, MEK1, p38α, p70S6K, PKA, PKCα, Src, and ZAP70) was performed using standard conditions (CEREP; www.cerep.com) using 200nM INCB018424. Significant inhibition was defined as more than or equal to 30% (average of duplicate assays) compared with control values.

Cell proliferation assay[1]
Cells were seeded at 2000/well of white bottom 96-well plates, treated with compounds from DMSO stocks (0.2% final DMSO concentration), and incubated for 48 hours at 37°C with 5% CO2. Viability was measured by cellular ATP determination using the Cell-Titer Glo luciferase reagent or viable cell counting. Values were transformed to percent inhibition relative to vehicle control, and IC50 curves were fitted according to nonlinear regression analysis of the data using PRISM GraphPad.

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Apoptosis[1]
Annexin V staining. Cells were treated for 20 to 24 hours and stained with annexin V and propidium iodide for analysis of early apoptotic and dead cells, respectively. Analysis was performed using a FACSCaliber flow cytometer. Mitochondrial membrane potential. Cells were treated for 24 hours and then incubated with 2μM of the dye JC-1. Analysis was performed by flow cytometry using 488-nm excitation and 530-nm and 585-nm emission filters. JC-1 exhibits potential-dependent accumulation in the mitochondria where its emission is in the red spectrum (590nM). A fluorescence shift from red (590nM) to green (530nM) indicates redistribution of the dye to the cytoplasm resulting from loss of mitochondrial membrane potential, an early marker for apoptosis.

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Colony-forming assay[1]
Mononuclear cells were isolated from peripheral blood from patients with PV or normal control persons by centrifugation through Ficoll. A total of 2 × 105 cells from control or patients with PV were plated onto methocult H88434 supplemented with recombinant cytokines (50 ng/mL stem cell factor, 10 ng/mL granulocyte-macrophage colony-stimulating factor, 10 ng/mL granulocyte colony-stimulating factor, 10 ng/mL IL-3, and 3 U/mL erythropoietin) and with indicated concentrations of INCB018424 or DMSO vehicle. For evaluation of endogenous erythroid colony growth, 3 to 4 × 105 cells from PV patients were plated onto minimal methocult medium with INCB018424 or vehicle. Each condition was performed in triplicate. Colonies derived from erythroid (burst-forming units [BFU] and colony-forming units [CFU]-E) and myeloid (CFU-granulocyte macrophage) progenitor cells were counted after 14 days.

JAK2V617F-driven mouse model
In vivo treatment with INCB018424 in a myeloproliferative neoplasm mouse model
All of the procedures were conducted in accordance with the US Public Health Service Policy on Humane Care and Use of Laboratory Animals. Mice were fed standard rodent chow and provided with water ad libitum. Ba/F3-JAK2V617F cells (105 per mouse) were inoculated intravenously into 6- to 8-week-old female BALB/c mice. Survival was monitored daily, and moribund mice were humanely killed and considered deceased at time of death. Treatment with vehicle (5% dimethyl acetamide, 0.5% methocellulose) or INCB018424 began within 24 hours of cell inoculation, twice daily by oral gavage. Hematologic parameters were measured using a Bayer Advia120 analyzed, and statistical significance was determined using Dunnett testing[1].

[1]. Quintas-Cardama A, et al. Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms. Blood, 2010, 115(15), 3109-3117.
[2]. Verstovsek S, et al. A double-blind, placebo-controlled trial of ruxolitinib for myelofibrosis. N Engl J Med, 2012, 366(9), 799-807.
[3]. Tavallai M, et al. Rationally Repurposing Ruxolitinib (Jakafi (®)) as a Solid Tumor Therapeutic.Front Oncol. 2016 Jun 13;6:14

Ruxolitinib is a pyrazole substituted at position 1 by a 2-cyano-1-cyclopentylethyl group and at position 3 by a pyrrolo[2,3-d]pyrimidin-4-yl group. Used as the phosphate salt for the treatment of patients with intermediate or high-risk myelofibrosis, including primary myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis. It has a role as an antineoplastic agent and an EC 2.7.10.2 (non-specific protein-tyrosine kinase) inhibitor. It is a nitrile, a pyrrolopyrimidine and a member of pyrazoles.

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Ruxolitinib, formerly known as INCB018424 or INC424, is an anticancer drug and a Janus kinase (JAK) inhibitor. It is a potent and selective inhibitor of JAK1 and JAK2, which are tyrosine kinases involved in cytokine signalling and hematopoiesis. Myeloproliferative neoplasms, such as myelofibrosis and polycythemia vera, are often characterized by aberrant activation of the JAK-STAT pathway, leading to abnormal blood cell counts and thrombotic complications. By inhibiting JAK1 and JAK2, ruxolitinib works to block the dysregulated cell signalling pathways and prevents abnormal blood cell proliferation. Due to a large number of patients with myeloproliferative neoplasms who have JAK2 mutations, ruxolitinib was the first ATP-competitive inhibitor of JAK1 and JAK2 ever developed. Ruxolitinib was first approved for the treatment of adult patients with myelofibrosis by the FDA in 2011, followed by EMA's approval in 2012. In 2014, it was approved for the treatment of polycythemia vera in adults who have an inadequate response to or are intolerant of [hydroxyurea] and in 2019, ruxolitinib was approved for use in steroid-refractory acute graft-versus-host disease in adults and children. The topical formulation of ruxolitinib is used to treat atopic dermatitis and vitiligo. It is being investigated for other inflammatory skin conditions. Ruxolitinib has been investigated to treat patients with coronavirus disease 2019 (COVID-19) accompanied by severe systemic hyperinflammation. In phase II clinical trials, ruxolitinib improved chest computed tomography and improved recovery in patients with lymphopenia. However, phase III clinical trials later determined that ruxolitinib was inadequate in meeting its primary endpoint of reducing the number of hospitalized COVID-19 patients who experienced severe complications thus the drug was not approved as a treatment for COVID-19.

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Ruxolitinib is a Kinase Inhibitor and Janus Kinase Inhibitor. The mechanism of action of ruxolitinib is as a Janus Kinase Inhibitor.
Ruxolitinib is a Kinase Inhibitor. The mechanism of action of ruxolitinib is as a Protein Kinase Inhibitor.
es Ruxolitinib is a small molecule Janus kinase inhibitor that is used in the treatment of intermediate or high risk myelofibrosis and resistant forms of polycythemia vera and graft-vs-host disease. Ruxolitinib is associated with transient and usually mild elevations in serum aminotransferase during therapy and to rare instances of self-limited, clinically apparent idiosyncratic acute liver injury as well as to cases of reactivation of hepatitis B in susceptible individuals.
Ruxolitinib is an orally bioavailable Janus-associated kinase (JAK) inhibitor with potential antineoplastic and immunomodulating activities. Ruxolitinib specifically binds to and inhibits protein tyrosine kinases JAK 1 and 2, which may lead to a reduction in inflammation and an inhibition of cellular proliferation. The JAK-STAT (signal transducer and activator of transcription) pathway plays a key role in the signaling of many cytokines and growth factors and is involved in cellular proliferation, growth, hematopoiesis, and the immune response; JAK kinases may be upregulated in inflammatory diseases, myeloproliferative disorders, and various malignancies.

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Ruxolitinib is indicated for the treatment of the following conditions: - intermediate or high-risk myelofibrosis (MF), including prima1y MF, post-polycythemia vera MF and post-essential thrombocythemia MF in adults. It is also used to treat disease-related splenomegaly or symptoms in adult patients with these conditions. - polycythemia vera (PV) in adults who have had an inadequate response to or are intolerant of hydroxyurea. - steroid-refracto1y acute graft-versus-host disease (GVHD) in adult and pediatric patients 12 years and older. - chronic GVHD in patients aged 12 years and older who have failed one or two lines of systemic therapy. Topical ruxolitinib is indicated for: - the short-term and non-continuous chronic treatment of mild to moderate atopic dermatitis in non-immunocompromised patients patients 12 years of age and older whose disease is not adequately controlled with topical prescription therapies or when those therapies are not advisable. - the treatment of non-segmental vitiligo in adult and pediatric patients 12 years of age and older.

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Ruxolitinib is an antineoplastic agent that inhibits cell proliferation, induces apoptosis of malignant cells, and reduces pro-inflammatory cytokine plasma levels by inhibiting JAK-induced phosphorylation of signal transducer and activator of transcription (STAT). Inhibition of STAT3 phosphorylation, which is used as a marker of JAK activity, by ruxolitinib is achieved at two hours after dosing which returned to near baseline by 10 hours in patients with myelofibrosis and polycythemia vera. In clinical trials, ruxolitinib reduced splenomegaly and improved symptoms of myelofibrosis. In a mouse model of myeloproliferative neoplasms, administration of ruxolitinib was associated with prolonged survival. Ruxolitinib inhibits both mutant and wild-type JAK2; however, JAK2V617F mutation, which is often seen in approximately 50% of patients with myelofibrosis, was shown to reduce ruxolitinib sensitivity, which may also be associated with possible resistance to JAK inhibitor treatment.

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Absorption: Following oral administration, ruxolitinib undergoes rapid absorption and the peak concentrations are reached within one hour after administration. Over a single-dose range of 5 mg to 200 mg, the mean maximal plasma concentration (Cmax) increases proportionally. Cmax ranged from 205 nM to 7100 nM and AUC ranged from 862 nM x hr to 30700 nM x hr. Tmax ranges from one to two hours following oral administration. Oral bioavailability is at least 95%.
Biological Half-Life: The mean elimination half-life of ruxolitinib is approximately 3 hours and the mean half-life of its metabolites is approximately 5.8 hours.

C17H18N6

306.3650

306.15930

C, 66.65; H, 5.92; N, 27.43

941678-49-5

Ruxolitinib (S enantiomer);941685-37-6;Ruxolitinib phosphate;1092939-17-7;(Rac)-Ruxolitinib-d9;2469553-67-9;Deuruxolitinib-d8;1513883-39-0;Ruxolitinib sulfate;1092939-16-6

25126798

Typically exists as white to light yellow solids at room temperature

1.4±0.1 g/cm3

592.6±50.0 °C at 760 mmHg

312.2±30.1 °C

0.0±1.7 mmHg at 25°C

1.747

1.69

83.18

N#CC[C@@H](N1N=CC(C2=C3C(NC=C3)=NC=N2)=C1)C4CCCC4

HFNKQEVNSGCOJV-OAHLLOKOSA-N

InChI=1S/C17H18N6/c18-7-5-15(12-3-1-2-4-12)23-10-13(9-22-23)16-14-6-8-19-17(14)21-11-20-16/h6,8-12,15H,1-5H2,(H,19,20,21)/t15-/m1/s1

(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile

INCB-018424, INCB 018424, INCB 18424, INCB-18424; INCB018424; INC424, INC424, INC-424; INCB18424, Jakafi and Jakavi (trade name)

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: 61 mg/mL (199.1 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility in Formulation 1: 5 mg/mL (16.32 mM) in 5% DMAC in 0.5% methylcellulose aqueous solution (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.79 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 3: ≥ 2.08 mg/mL (6.79 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 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 4: ≥ 2.08 mg/mL (6.79 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 corn oil and mix evenly.

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Solubility in Formulation 5: 2% DMSO+30% PEG 300+ddH2O:5mg/mL

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Solubility in Formulation 6: 5 mg/mL (16.32 mM) in 0.5% Methylcellulose/saline water (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
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.)