Tie-2 (IC50 = 1 nM); p38α (IC50 = 35 nM); p38β (IC50 = 26 nM)

Pexmetinib is a dual inhibitor of Tie-2 and p38 MAPK, with IC50 values for Tie-2, p38α and p38β of 1 nM, 35 nM, and 26 nM, respectively. The IC50 values for pexmetinib are also 4 nM for Abl, 10 nM for Arg, 28 nM for FGFR1, 47 nM for Flt1, 42 nM for Flt4, 41 nM for Fyn, 26 nM for Hck, 25 nM for Lyn, and 26 nM for MINK, respectively. In myelodysplastic syndromes, pexmetinib (0.5, 1 μM) inhibits leukemic cell proliferation and promotes hematopoietic activity.

Pexmetinib (30 mg/kg, p.o.) prevents the production of pro-inflammatory cytokines TNF and IL6 in male Swiss Webster mice in response to lipopolysaccharide (LPS) or staphylococcus enterotoxin A. ARRY-614 (25 mg/kg, p.o.) inhibits tumor growth in established RPMI 8226 xenografts and exhibits additive activity when combined with thalidomide. When combined with Taxol, ARRY-614 (30 mg/kg, p.o.) exhibits additive tumor growth inhibition activity in ovarian carcinoma A2780 xenografts.

Pexmetinib (ARRY-614) is a potent inhibitor of cytokine synthesis, via the dual inhibition of p38 mitogen-activated protein kinase (MAPK), and Tie2/Tek receptor tyrosine kinase. The in vitro IC50 values of ARR Y-614 for both Tie2 and p38 mitogen-activated protein kinase are 1000 ng/mL and 100 ng/mL, respectively.

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Correction for protein binding [1]
LPS-induced TNFα was determined in undiluted whole blood (WB) and peripheral blood mononuclear cells (PBMCs) from matched donors (N=9 healthy human subjects). Blood was collected by venipuncture into heparinized tubes for WB samples and into CPT tubes for PBMCs. PBMCs were isolated according to manufacturer's directions and resuspended at 1 million/mL in RPMI-1640 supplemented with 2% heat-inactivated fetal bovine serum. WB and PBMC's were pre-incubated with varying concentrations of Pexmetinib for 1 hr, 37°C, 5% CO2 then stimulated with 100 ng/ml LPS for 16 hours at 37°C, 5% CO2. TNF-α levels in the cell-free supernatants were quantified by ELISA as a measure of p-p38 inhibition.

Pexmetinib, an inhibitor of TIE2/p38, is incubated with cell lines and primary samples at the recommended doses. Viability is measured using a Fluostar Omega Microplate Reader and Cell Titer Blue to determine viability[1].

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In vitro assays for determination of IC50 [1]
Human Umbilical Vein Endothelial Cells (HUVEC) and HEK-293 human embryonic kidney cells engineered to express constitutively active Tie-2 (HEK-Tie2) were used to assess p38 and Tie2 phosphorylation in vitro. To create HEK-Tie2 cells, a synthetic cDNA was generated that directs the synthesis of a hybrid form of the Tie-2 receptor engineered to contain a FLAG epitope tag just downstream of a prolactin signal peptide at the extreme amino terminus of the polypeptide. In addition, this receptor construct was engineered with an arginine to tryptophan substitution at amino acid position 849 which has confers constitutive activation of the receptor. The cDNA was subcloned into the retroviral vector pLNCX2 using the HindIII and NotI sites in the polylinker and contains a doxycyclin-inducible promoter. HEK-Tie2 cells were treated with 1 μg/mL doxycyclin 24 hours prior to drug treatment to induce Tie-2 expression, and HUVECs were pretreated for 1 hr with 1 μg/mL anisomycin or 0.1 μg/mL recombinant angpt-1 to activate p38 or Tie-2, respectively. Cells were treated with varying concentrations of Pexmetinib for 2hr (0.25% BSA, 0.2% DMSO), lysed in RIPA buffer then subjected to Western blot analysis. Effect on phosphorylation was detected by near-infrared(NIR) fluorescence using primary antibodies directed to p-p38, pHsp27, pTie2 or pAkt followed by infrared labeled secondary antibodies Alexa Fluor 680 donkey anti-rabbit or donkey anti-mouse IR800, normalized to GAPDH using LICOR Odyssey software.

Male Swiss Webster mice
30 mg/kg
p.o.

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In vivo assays for target inhibition of p-p38 and pTie2 in vivo [1]
Naïve male CD-1 (Charles River) or female nu/nu NCr mice inoculated with 5×10^6 HEK-Tie2 cells subcutaneously near the axillary region on the right flank were utilized to assess the relationship between plasma concentration and target inhibition in lung (p-p38) or tumor (p-p38 and pTie2). To induce Tie2 expression in tumor-bearing animals, a single dose of 30 mg/kg doxycycline in 5% sucrose was administered 16 hours prior to administration of Pexmetinib (amorphous free base) as a suspension in 1% CMC/0.02%SDS. All treatments were administered by oral gavage in a dosing volume of 10 mL/kg. At predetermined time points, plasma was collected for analysis of drug concentrations by LC-MS/MS and tissue (lung or tumor) harvested for analysis of target inhibition by Western blot. Lung and tumor tissue were homogenized in RIPA buffer then subjected to immunoblot analysis. Effect on phosphorylation was detected by near-infrared (NIR) fluorescence as above, normalized to GAPDH and data analyzed relative to vehicle-treated control.

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Functional inhibition of p-p38 in vivo [1]
The lipopolysaccharide (LPS) challenge or endotoxemia model evaluates the ability of an animal to mount an acute phase response to an inflammatory stimulus. A hallmark of this response is production of TNFα which is mediated by p38 pathway activation, thus, inhibition of TNFα provides a functional readout of p38 inhibition. Naïve Swiss Webster mice were orally administered increasing doses of Pexmetinib as a single agent 30 minutes prior to intraperitoneal challenge with 2 mg/kg LPS. Ninety minutes after LPS injection, whole blood was collected to process for serum and TNFα measured by ELISA.

[1]. Pexmetinib: A Novel Dual Inhibitor of Tie2 and p38 MAPK with Efficacy in Preclinical Models of Myelodysplastic Syndromes and Acute Myeloid Leukemia. Cancer Res. 2016 Aug 15;76(16):4841-4849.

Pexmetinib is under investigation in clinical trial NCT04074967 (Study of ARRY-614 Plus Either Nivolumab or Ipilimumab).
Pexmetinib is an orally bioavailable small-molecule inhibitor of p38 and Tie2 kinases with potential antineoplastic, anti-inflammatory and antiangiogenic activities. Pexmetinib binds to and inhibits the activities of p38 and Tie2 kinases, which may inhibit the production of proinflammatory cytokines and may decrease tumor angiogenesis and tumor cell growth and survival. p38 is a MAP kinase that is often upregulated in cancer cells, playing a crucial part in the production of a variety of cytokines involved in inflammation and cellular proliferation such as tumor necrosis factor (TNF) and interleukin (IL)-1 and -6. Tie2 is an endothelial cell specific receptor that is activated by angiopoietins, growth factors required for angiogenesis. This agent has also been reported to inhibit other kinases including vascular endothelial growth factor receptor (VEGFR2) and Src tyrosine kinases.

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Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) suppress normal hematopoietic activity in part by enabling a pathogenic inflammatory milieu in the bone marrow. In this report, we show that elevation of angiopoietin-1 in myelodysplastic CD34(+) stem-like cells is associated with higher risk disease and reduced overall survival in MDS and AML patients. Increased angiopoietin-1 expression was associated with a transcriptomic signature similar to known MDS/AML stem-like cell profiles. In seeking a small-molecule inhibitor of this pathway, we discovered and validated pexmetinib (ARRY-614), an inhibitor of the angiopoietin-1 receptor Tie-2, which was also found to inhibit the proinflammatory kinase p38 MAPK (which is overactivated in MDS). Pexmetinib inhibited leukemic proliferation, prevented activation of downstream effector kinases, and abrogated the effects of TNFα on healthy hematopoietic stem cells. Notably, treatment of primary MDS specimens with this compound stimulated hematopoiesis. Our results provide preclinical proof of concept for pexmetinib as a Tie-2/p38 MAPK dual inhibitor applicable to the treatment of MDS/AML. [1]

C31H33FN6O3

556.63

556.259

C, 66.89; H, 5.98; F, 3.41; N, 15.10; O, 8.62

945614-12-0

24765037

White to off-white solid powder

1.3±0.1 g/cm3

694.1±55.0 °C at 760 mmHg

373.6±31.5 °C

0.0±2.3 mmHg at 25°C

1.635

6.81

3

6

9

41

852

0

O=C(NCC1C(OC2C=C3C=NN(C3=CC=2)CCO)=CC=C(F)C=1)NC1N(C2C=CC(C)=CC=2)N=C(C(C)(C)C)C=1

LNMRSSIMGCDUTP-UHFFFAOYSA-N

InChI=1S/C31H33FN6O3/c1-20-5-8-24(9-6-20)38-29(17-28(36-38)31(2,3)4)35-30(40)33-18-22-15-23(32)7-12-27(22)41-25-10-11-26-21(16-25)19-34-37(26)13-14-39/h5-12,15-17,19,39H,13-14,18H2,1-4H3,(H2,33,35,40)

1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[[5-fluoro-2-[1-(2-hydroxyethyl)indazol-5-yl]oxyphenyl]methyl]urea

ARRY 614; ARRY614; Pexmetinib [INN]; Pexmetinib (ARRY-614); UNII-3750D0U8B5; 1-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(5-fluoro-2-((1-(2-hydroxyethyl)-1H-indazol-5-yl)oxy)benzyl)urea; 1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[[5-fluoro-2-[1-(2-hydroxyethyl)indazol-5-yl]oxyphenyl]methyl]urea; ARRY-614; Pexmetinib

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: ≥ 2.08 mg/mL (3.74 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 2: ≥ 2.08 mg/mL (3.74 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 3: ≥ 2.08 mg/mL (3.74 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.)