ERK1 ; ERK2 (IC50 = 0.3 nM)

Ulixertinib lowers the levels of 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. Additionally, ulixertinib reduces A375 cell proliferation with an IC50 of 180 nM. [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 M 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.

Cell media containing 10% (v/v) foetal calf serum and 1% (v/v) L-glutamine are used to cultivate A375 cells. Cells are harvested, dispensed into black, 384-well Costar plates with a capacity of 40 L cell media and 200 cells per well, 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 dosed directly into the cell plates' inner 308 wells. To determine compound IC50s, the cells are dosed over a 12 point range from 30 M to 0.03 nM, with a final DMSO concentration in the assay of 0.3%. The cell plates are then kept at 37°C for the following 72 hours. After 30 minutes of incubation at room temperature, cells were fixed and stained by adding 20 µL of 12% formaldehyde to PBS/A (for a final concentration of 4%), along with a 1:2000 dilution of Hoechst 33342. Using a Cellomics ArrayScanTM VTI imaging platform, a cell count is carried out on the stained cell plates. Additionally, a Day 0 cell plate is fixed, stained, and read to produce a baseline cell count for calculating the compound's cytotoxic and anti-proliferative effects.

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 a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and ERK1/2 selectivity. It is currently in clinical trials for the treatment of a wide range of tumors.
Ulixertinib is an orally available inhibitor of extracellular signal-regulated kinase (ERK) 1 and 2, with potential antineoplastic activity. Upon oral administration, ulixertinib inhibits both ERK 1 and 2, thereby preventing the activation of ERK-mediated signal transduction pathways. This results in the inhibition of ERK-dependent tumor cell proliferation and survival. The mitogen-activated protein kinase (MAPK)/ERK pathway is often upregulated in a variety of tumor cell types and plays a key role in tumor cell proliferation, differentiation and survival.

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The RAS/RAF/MEK/ERK signaling pathway has been targeted with a number of small molecule inhibitors in oncology clinical development across multiple disease indications. Importantly, cell lines with acquired resistance to B-RAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition by small molecule inhibitors. There are a number of selective, noncovalent ERK1/2 inhibitors reported along with the promiscuous hypothemycin (and related analogues) that act via a covalent mechanism of action. This article reports the identification of multiple series of highly selective covalent ERK1/2 inhibitors informed by structure-based drug design (SBDD). As a starting point for these covalent inhibitors, reported ERK1/2 inhibitors and a chemical series identified via high-throughput screening were exploited. These approaches resulted in the identification of selective covalent tool compounds for potential in vitro and in vivo studies to assess the risks and or benefits of targeting this pathway through such a mechanism of action. [1]

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Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease in urgent need of newer therapeutic modalities. Majority of patients with PDAC have mutations in KRAS, which unfortunately remains an ineffectual target. Our strategy here is to target KRAS downstream effectors PI3K and mTOR. In this study, we investigated the antitumor efficacy of the novel PI3K and mTOR dual inhibitor VS-5584 in PDAC. Our data shows that PI3K/mTOR dual inhibition causes ERK activation in all tested PDAC cell lines. Although the MEK inhibitor GSK1120212 could abrogate VS-5584-induced ERK activation, it did not substantially enhance cell death in all the cell lines tested. However, combination with ERK inhibitor SCH772984 not only mitigated VS-5584-induced ERK activation but also enhanced VS-5584-induced cell death. In a xenograft model of PDAC, we observed 28% and 44% tumor inhibition for individual treatment with VS-5584 and SCH772984, respectively, while the combined treatment showed superior tumor inhibition (80%) compared to vehicle control treatment. Our findings support the clinical development of VS-5584 and ERK inhibitor combination for PDAC treatment. [3]

C21H22CL2N4O2

433.33

432.111

C, 58.21; H, 5.12; Cl, 16.36; N, 12.93; O, 7.38

869886-67-9

Ulixertinib hydrochloride;1956366-10-1

11719003

White to off-white solid powder

1.4±0.1 g/cm3

682.8±55.0 °C at 760 mmHg

366.8±31.5 °C

0.0±2.2 mmHg at 25°C

1.650

5.16

4

4

7

29

539

1

ClC1C=NC(NC(C)C)=CC=1C1=CNC(C(=O)N[C@@H](C2C=CC=C(Cl)C=2)CO)=C1

KSERXGMCDHOLSS-LJQANCHMSA-N

InChI=1S/C21H22Cl2N4O2/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)/t19-/m1/s1

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

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: ≥ 2.5 mg/mL (5.77 mM) 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 2: ≥ 2.5 mg/mL (5.77 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.77 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 4: ≥ 2.5 mg/mL (5.77 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 5: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 4.3mg/ml

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Solubility in Formulation 6: 10 mg/mL (23.08 mM) in 1% (w/v) carboxymethylcellulose (CMC) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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