ERK1; ERK2 (IC50 <0.3 nM)

Ulixertinib decreases phosphorylated ERK2 (pERK) and phosphorylation of the downstream kinase RSK (pRSK) in the A375 melanoma cell line, which has the b-RAFV600E mutation, with IC50 values of 4.1/0.14 μM, respectively. With an IC50 of 180 nM, ulixertinib also prevents A375 cell proliferation. [1]
Ulixertinib (BVD-523, VRT752271) is a novel small molecule, which potently and selectively inhibits ERK1 and ERK2 kinases in a reversible ATP-competitive fashion [2].

In the pharmacokinetic study, the sensitivity and specificity of the assay are found to be sufficient for accurately characterizing the plasma pharmacokinetics of Ulixertinib (VRT752271) in Balb/C mice.

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Ulixertinib inhibits tumor growth in vivo in BRAF-mutant melanoma and colorectal xenografts as well as in KRAS-mutant colorectal and pancreatic models. In clinical studies, ulixertinib was well tolerated by patients with advanced solid tumors. In an oral Phase-I dose escalation study (having 9 doses) with an end point to determine the dose limited toxicities (DLT), maximum tolerated dose (MTD) along with pharmacokinetic profile and preliminary efficacy assessment it was administered in a dose range of 10–900 mg in a b.i.d regimen. Ulixertinib showed linear pharmacokinetics up to 600 mg (b.i.d), this was found to be MTD. [2]

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A sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for the quantification of ulixertinib in mice plasma using phenacetin as an internal standard (I.S.) as per regulatory guidelines. Sample preparation was accomplished through a protein precipitation procedure with acetonitrile:methanol mixture. Chromatographic separation was performed on Atlantis dC18 column using a binary gradient using mobile phase A (0.2% formic acid in water) and B (acetonitrile) at a flow rate of 0.60mL/min. Elution of ulixertinib and I.S. occurred at ∼1.07 and 1.20min, respectively. The total chromatographic run time was 2.5min. A linear response function was established in the concentration range of 1.58-2054ng/mL. The intra- and inter-day accuracy and precisions were in the range of 2.11-11.8 and 5.80-11.4%, respectively. This novel method has been applied to a pharmacokinetic study in mice[2].

MEK U911-activated ERK2 protein is expressed and purified in-house. Enzyme and substrate solutions are prepared in assay buffer, which is composed of 50 mM Tris (pH 7.5), 10 mM MgCl2, 0.1 mM EGTA, 10 mM DTT, and 0.01% (v/v) CHAPS. A polypropylene, 384-well plate with test and reference control substances is filled with 10 µL of 1.2 nM ERK2 protein that has been prepared in assay buffer. In order to determine the compound IC50s, the compound plates had previously been dosed with a 12-point range from 100 µM down to 0.1 nM, with a total DMSO concentration in the assay of 1%. Following a 20-minute pre-incubation period at room temperature, 10 µL of substrate solution—consisting of 16 µM erktide (IPTTPITTTYFFFK) and 120 µM ATP (measured Km) in assay buffer—is added. The addition of 80 µl of 1% (v/v) formic acid quenches the reaction after it has been allowed to proceed for 20 minutes at room temperature. The RapidFire Mass Spectrometry platform is then used to run the assay plates in order to measure the substrate (unphosphorylated Erktide) and product (phosphorylated Erktide) levels.

A375 cells are cultured in cell media composed of DMEM, 10% (v/v) Foetal Calf Serum and 1% (v/v) L-Glutamine. Cells are harvested, dispensed into black 384-well Costar plates with 200 cells per well and a total volume of 40 L cell media, and then incubated overnight at 37°C, 90% relative humidity, and 5% CO2 in a rotating incubator. Using a Labcyte Echo 555 acoustic dispenser, test substances and reference controls are directly dosed into the inner 308 wells of the cell plates. With a total DMSO concentration in the assay of 0.3%, the cells are dosed over a 12 point range from 30 M down to 0.03 nM in order to determine the compound IC50s. Following that, the cell plates are incubated for 72 hours at 37°C. In order to fix and stain the cells, 20 µL of 12% formaldehyde was added to PBS/A (4% final concentration) along with a 1:2000 dilution of Hoechst 33342. The cells were then incubated for 30 minutes at room temperature before being washed with PBS/A. A Cellomics ArrayScanTM VTI imaging platform is used to count the cells on the stained cell plates. Additionally, a Day 0 cell plate is fixed, stained, and read to produce a baseline cell count for assessing both the anti-proliferative and cytotoxic effects of the compound.

Pharmacokinetic study [2]
Male Balb/C mice (n = 24) were housed in Jubilant Biosys animal house facility at 22 ± 2 °C and at humidity (30–70%) controlled room (15 air changes/h) with a 12:12 h light:dark cycles, had free access to rodent feed and water for one week before using for experimental purpose. Following ∼4 h fast (during the fasting period animals had free access to water) animals were divided into two groups (n = 12/group). Group I animals (25–28 g) received Ulixertinib orally at 10 mg/kg (strength: 1.0 mg/mL; dose volume: 10 mL/kg), whereas Group II animals (29–31 g) received Ulixertinib intravenously (strength: 0.1 mg/mL; dose volume: 10 mL/kg) at 1.0 mg/kg dose. Post-dosing serial blood samples (50 μL, sparse sampling was done and at each time point three mice were used for blood sampling) were collected using Micropipettes through tail vein into polypropylene tubes containing Na2·EDTA solution as an anti-coagulant at 0.25, 0.5, 1, 2, 4, 8, 10 and 24 (for oral study) and 0.12, 0.25, 0.5, 1, 2, 4, 8 and 24 (for intravenous study). Plasma was harvested by centrifuging the blood using Biofuge at 1760 g for 5 min and stored frozen at −80 ± 10 °C until analysis. Animals were allowed to access feed 2 h post-dosing.
The criteria for acceptance of the analytical runs encompassed the following: (i) 67% of the QC samples accuracy must be within 85–115% of the nominal concentration (ii) not less than 50% at each QC concentration level must meet the acceptance criteria. Plasma concentration-time data of Ulixertinib was analyzed by non-compartmental method using Phoenix WinNonlin Version 6.3.

[1]. Structure-Guided Design of Highly Selective and Potent Covalent Inhibitors of ERK1/2. J Med Chem. 2015 Jun 11;58(11):4790-801.

[2]. Determination of ulixertinib in mice plasma by LC-MS/MS and its application to a pharmacokinetic study in mice. J Pharm Biomed Anal. 2016 Jun 5;125:140-4.

[3]. Targeting ERK Enhances the Cytotoxic Effect of the Novel PI3K and mTOR Dual Inhibitor VS-5584 in Preclinical Models of Pancreatic Cancer. Oncotarget. 2017 Jul 4;8(27):44295-44311.

Ulixertinib is a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and ERK1/2 selectivity. It is currently undergoing clinical trials for the treatment of various cancers. Ulixertinib is an orally administered extracellular signal-regulated kinase (ERK) 1 and 2 inhibitor with potential antitumor activity. After oral administration, Ulixertinib simultaneously inhibits ERK1 and 2, thereby preventing the activation of ERK-mediated signal transduction pathways. This leads to the inhibition of ERK-dependent tumor cell proliferation and survival. The mitogen-activated protein kinase (MAPK)/ERK pathway is frequently upregulated in various tumor cell types and plays a crucial role in tumor cell proliferation, differentiation, and survival. The RAS/RAF/MEK/ERK signaling pathway has become a target for various small molecule inhibitors in the clinical development of oncology for multiple disease indications. Importantly, cell lines that have been shown to develop resistance to B-RAF and MEK inhibitors still remain sensitive to small molecule ERK1/2 inhibitors. Currently, a variety of selective non-covalent ERK1/2 inhibitors and broad-spectrum ERK1/2 inhibitors with covalent mechanisms of action (such as hypothemycin and its analogues) have been reported. This article reports a series of highly selective covalent ERK1/2 inhibitors identified using a structural drug design (SBDD) approach. The design of these covalent inhibitors is based on the reported ERK1/2 inhibitors and a series of compounds identified through high-throughput screening. These methods ultimately identify a number of selective covalent tool compounds that can be used in potential in vitro and in vivo studies to assess the risks and/or benefits of targeting this pathway through this mechanism of action. [1]

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Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that urgently requires new treatments. Most patients with pancreatic ductal adenocarcinoma (PDAC) have KRAS gene mutations, but unfortunately, KRAS remains a poorly treated target. Our strategy is to target the downstream effector molecules PI3K and mTOR of KRAS. This study investigates the antitumor efficacy of the novel PI3K/mTOR dual inhibitor VS-5584 in PDAC. Our data showed that dual inhibition of PI3K/mTOR activated ERK in all tested PDAC cell lines. Although the MEK inhibitor GSK1120212 inhibited VS-5584-induced ERK activation, it did not significantly enhance cell death in all tested cell lines. However, the combination of GSK1120212 and the ERK inhibitor SCH772984 not only alleviated VS-5584-induced ERK activation but also enhanced VS-5584-induced cell death. In a pancreatic ductal adenocarcinoma (PDAC) xenograft model, we observed that treatment with VS-5584 and SCH772984 alone inhibited tumor growth by 28% and 44%, respectively, while the combination therapy showed a superior tumor inhibition rate (80%) compared to the vector control group. Our results support the clinical development of VS-5584 in combination with an ERK inhibitor for the treatment of PDAC. [3]

C21H23CL3N4O2

469.792

468.088

C, 53.69; H, 4.93; Cl, 22.64; N, 11.93; O, 6.81

1956366-10-1

Ulixertinib;869886-67-9

71584481

White to off-white solid powder

5

4

7

30

539

1

CC(C)NC1=NC=C(C(=C1)C2=CNC(=C2)C(=O)N[C@H](CO)C3=CC(=CC=C3)Cl)Cl.Cl

DKGYQCPFBWFTHM-FSRHSHDFSA-N

InChI=1S/C21H22Cl2N4O2.ClH/c1-12(2)26-20-8-16(17(23)10-25-20)14-7-18(24-9-14)21(29)27-19(11-28)13-4-3-5-15(22)6-13;/h3-10,12,19,24,28H,11H2,1-2H3,(H,25,26)(H,27,29);1H/t19-;/m1./s1

N-[(1S)-1-(3-chlorophenyl)-2-hydroxyethyl]-4-[5-chloro-2-(propan-2-ylamino)pyridin-4-yl]-1H-pyrrole-2-carboxamide;hydrochloride

BVD-523; VRT752271BVD-523; Ulixertinib hydrochloride; 1956366-10-1; Ulixertinib HCl; Ulixertinib (hydrochloride); 3K792HRQ8B; BVD 523; BVD523; VRT752271; VRT752271; VRT 752271; Ulixertinib

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, 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)

DMSO: 86~100 mg/mL (198.5~212.9 mM)
Ethanol: ~86 mg/mL (~198.5 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.43 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 (4.43 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 (4.43 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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Solubility in Formulation 4: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 4.3mg/ml

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