MEK1 (IC50 = 5.2 nM); MEK2 (IC50 = 5.2 nM)

RO4987655 potently inhibits mitogen-activated protein kinase signaling pathway activation and tumor cell growth, with an in vitro IC50 of 5.2 nM for inhibition of MEK1/2[1]. With an IC50 value of 0.0065 μM, RO4987655 inhibits the proliferation of NCI-H2122 cells in a dose-dependent manner. At doses ranging from 0.1 to 1.0 μM, RO4987655 suppresses pERK1/2 as early as 2 hours into the treatment period[2].

In xenograft models, RO4987655 (CH4987655) administered orally as a single agent completely eradicates tumors. With a tmax of under one hour, RO4987655 is rapidly absorbed. From 0.5 to 4 mg, exposures are dose-proportional. With a terminal t1/2 of under 25 hours, the disposition is biphasic. Low intersubject variability is observed; the range for Cmax and area-under-the-curve (AUC) is respectively 9%–23% and 14%–25%. At higher doses, pERK inhibition is more than 80% inhibited and exposure dependent. An inhibitory Emax model (Emax ~100%; IC50 40.6 ng/mL) is used in nonlinear mixed-effect modeling to describe the pharmacokinetic-pharmacodynamic relationship[1]. Randomly assigned study groups are made up of female athymic nude mice. Using a digital caliper and on days 0, 1, and 3 with doses of 1.0, 2.5, and 5.0 mg/kg RO4987655, the tumor size is estimated. Over this period, the vehicle treatment does not stop the NCI-H2122 tumor xenograft from growing. On the other hand, treatment with RO4987655 causes tumor growth inhibition (TGI) of 119% at 1.0 mg/kg, 145% at 2.5 mg/kg, and 150% at 5.0 mg/kg on day 3. PET imaging demonstrates that [18F] FDG uptake in the xenografts decreases 24 hours (day 1) after RO4987655 administration[2].

RO4987655 (also known as CH-4987655) is a novel, orally bioavailable and specific small molecule inhibitor of MEK kinase with an IC50 of 5.2 nM for MEK1/MEK2. Mitogen-activated protein kinase kinase 1 (MAP2K1/MEK1), which may have antineoplastic activity, is the target of this medication. With an in vitro IC50 for MEK1/2 inhibition of 5.2 nmol/L, it effectively prevents the activation of the mitogen-activated protein kinase signaling pathway and the growth of tumor cells.

The heat-inactivated fetal bovine serum and L-glutamine are maintained at the indicated concentrations in the designated media for the human lung adenocarcinoma cell line NCI-H2122. At 37 degrees Celsius and 5% CO2, cells develop. Viable cells were counted using the Cell Counting Kit-8 after cells were exposed to RO4987655 at different concentrations (0.00001, 0.001, 0.1, and 10 μM) for 72 hours in 96-well plates[2].

Mice: Mice that are athymic and naked in females It uses balb nu/nu that are 5 to 6 weeks old (18 to 22 g). Balb-nu/nu mice receive a subcutaneous injection of NCI-H2122 cells ((4×106/mouse). Mice are randomized into groups with comparable mean tumor volumes at the beginning of the study once tumors are established (100 to 200 mm3). On days 0, 1, and 3 with doses of 1.0, 2.5, and 5.0 mg/kg RO4987655, PET scans are used to estimate the tumor size. Days 0 (baseline), 1, 2, 3, and 9 of [18F] FDG-PET imaging are used to measure tumor volume and body weight. Calculations are made to determine tumor growth inhibition[2].

Purpose: CH4987655 (RO4987655) is an orally active and highly selective small-molecule MEK inhibitor. It potently inhibits mitogen-activated protein kinase signaling pathway activation and tumor cell growth, with an in vitro IC(50) of 5.2 nmol/L for inhibition of MEK1/2. Single-agent oral administration of CH4987655 resulted in complete tumor regressions in xenograft models. [1]
Experimental design: All 40 subjects received a single oral dose followed by 72 hrs of pharmacokinetic, pharmacodynamic, and safety/tolerability assessments. The pharmacodynamics were measured by changes in phosphorylated extracellular signal-regulated kinase (pERK) levels in a surrogate tissue peripheral blood mononuclear cells ex vivo stimulated by PMA. [1]
Results: Doses of 0.5, 1, 2, 3, and 4 mg were safe and well tolerated. No clinically significant safety event was observed. A total of 26 adverse events (n = 15) were reported: 21 mild, 5 moderate, and none severe. Moderate adverse events were experienced by one subject at 1 mg (autonomic nervous system imbalance) and three subjects at 4 mg (diarrhea, abdominal pain, autonomic nervous system and acne). CH4987655 was rapidly absorbed with a t(max) of approximately 1 h. Exposures were dose proportional from 0.5 to 4 mg. The disposition was biphasic with a terminal t(1/2) of approximately 25 hr. Intersubject variability was low, 9% to 23% for C(max) and 14% to 25% for area-under-the-curve (AUC). pERK inhibition was exposure dependent and was greater than 80% inhibition at higher doses. The pharmacokinetic-pharmacodynamic relationship was characterized by an inhibitory E(max) model (E(max) approximately 100%; IC(50) 40.6 ng/mL) using nonlinear mixed-effect modeling. [1]
Conclusions: A significant extent of pERK inhibition was achieved for a single dose that was considered to be safe and well tolerated in healthy volunteers.

[1]. The safety, tolerability, pharmacokinetics, and pharmacodynamics of single oral doses of CH4987655 in healthy volunteers: target suppression using a biomarker. Clin Cancer Res. 2009 Dec 1;15(23):7368-74.

[2]. Tegnebratt T, et al. Evaluation of efficacy of a new MEK inhibitor, RO4987655, in human tumor xenografts by [(18)F] FDG-PET imaging combined with proteomic approaches. EJNMMI Res. 2014 Dec;4(1):34.

RO4987655 has been used in trials studying the treatment of Neoplasms.
MEK Inhibitor RO4987655 is an orally active small molecule, targeting mitogen-activated protein kinase kinase 1 (MAP2K1 or MEK1), with potential antineoplastic activity. MEK inhibitor RO4987655 binds to and inhibits MEK, which may result in the inhibition of MEK-dependent cell signaling and the inhibition of tumor cell proliferation. MEK, a dual specificity threonine/tyrosine kinase, is a key component of the RAS/RAF/MEK/ERK signaling pathway that regulates cell growth; constitutive activation of this pathway has been implicated in many cancers.

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Inhibition of mitogen-activated protein kinase (MEK, also known as MAPK2, MAPKK), a key molecule of the Ras/MAPK (mitogen-activated protein kinase) pathway, has shown promising effects on B-raf-mutated and some RAS (rat sarcoma)-activated tumors in clinical trials. The objective of this study is to examine the efficacy of a novel allosteric MEK inhibitor RO4987655 in K-ras-mutated human tumor xenograft models using [(18)F] FDG-PET imaging and proteomics technology. Methods: [(18)F] FDG uptake was studied in human lung carcinoma xenografts from day 0 to day 9 of RO4987655 therapy using microPET Focus 120 (CTI Concorde Microsystems, Knoxville, TN, USA). The expression levels of GLUT1 and hexokinase 1 were examined using semi-quantitative fluorescent immunohistochemistry (fIHC). The in vivo effects of RO4987655 on MAPK/PI3K pathway components were assessed by reverse phase protein arrays (RPPA). Results: We have observed modest metabolic decreases in tumor [(18)F] FDG uptake after MEK inhibition by RO4987655 as early as 2 h post-treatment. The greatest [(18)F] FDG decreases were found on day 1, followed by a rebound in [(18)F] FDG uptake on day 3 in parallel with decreasing tumor volumes. Molecular analysis of the tumors by fIHC did not reveal statistically significant correlations of GLUT1 and hexokinase 1 expressions with the [(18)F] FDG changes. RPPA signaling response profiling revealed not only down-regulation of pERK1/2, pMKK4, and pmTOR on day 1 after RO4987655 treatment but also significant up-regulation of pMEK1/2, pMEK2, pC-RAF, and pAKT on day 3. The up-regulation of these markers is interpreted to be indicative of a reactivation of the MAPK and activation of the compensatory PI3K pathway, which can also explain the rebound in [(18)F] FDG uptake following MEK inhibition with RO4987655 in the K-ras-mutated human tumor xenografts. Conclusions: We have performed the first preclinical evaluation of a new MEK inhibitor, RO4987655, using a combination of [(18)F] FDG-PET imaging and molecular proteomics. These results provide support for using preclinical [(18)F] FDG-PET imaging in early, non-invasive monitoring of the effects of MEK and perhaps other Ras/MAPK signaling pathway inhibitors, which should facilitate a wider implementation of clinical [(18)F] FDG-PET to optimize their clinical use.[2]

C20H19F3IN3O5

565.28

565.032

C, 42.49; H, 3.39; F, 10.08; I, 22.45; N, 7.43; O, 14.15

874101-00-5

11548630

White to off-white solid powder

1.7±0.1 g/cm3

1.638

5.49

3

9

8

32

652

0

O=C(C1C(NC2C(F)=CC(I)=CC=2)=C(F)C(F)=C(CN2C(=O)CCCO2)C=1)NOCCO

FIMYFEGKMOCQKT-UHFFFAOYSA-N

InChI=1S/C20H19F3IN3O5/c21-14-9-12(24)3-4-15(14)25-19-13(20(30)26-31-7-5-28)8-11(17(22)18(19)23)10-27-16(29)2-1-6-32-27/h3-4,8-9,25,28H,1-2,5-7,10H2,(H,26,30)

3,4-difluoro-2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-5-[(3-oxooxazinan-2-yl)methyl]benzamide

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 (4.42 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 (4.42 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 (4.42 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 4: 10% DMSO+ 40% PEG300+ 5% Tween-80+ 45% saline: ≥ 2.5 mg/mL

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