MEK1 (IC50 = 5.2 nM); MEK2 (IC50 = 5.2 nM)
MEK1/2 (IC₅₀ = 5.2 nmol/L for inhibition of MEK1/2 in vitro) [1]
- MEK1/2 (IC₅₀ = 0.0065 μM for anti-proliferative inhibition in NCI-H2122 cells) [2]

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].

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1. RO4987655 (also named CH4987655) is an orally active and highly selective small-molecule MEK inhibitor, which potently inhibits mitogen-activated protein kinase (MAPK) signaling pathway activation and tumor cell growth in vitro, with an IC₅₀ of 5.2 nmol/L for the inhibition of MEK1/2 [1]
2. In NCI-H2122 (K-ras-mutated) human lung carcinoma cells treated with RO4987655 at indicated concentrations (0, 0.01, 0.03, 0.1, 0.3, 1 μM) for 2 hours, immunoblotting analysis of cell lysates showed that RO4987655 inhibited the phosphorylation of ERK1/2 (pERK1/2) in a concentration-dependent manner; the phosphorylation levels of MEK1/2 (pMEK1/2) and AKT (pAKT) also changed with the increase of drug concentration. In time-dependent experiments, NCI-H2122 cells treated with 0.1 μM RO4987655 for 0, 6 hours, day 1 (D1), day 2 (D2), day 3 (D3) showed dynamic changes in the phosphorylation levels of ERK, MEK, AKT and EGFR: pERK was down-regulated at early time points, while pMEK1/2, pMEK2, pC-RAF and pAKT were up-regulated on day 3. The dose-dependent anti-proliferative effect of RO4987655 in NCI-H2122 cells was confirmed, with an IC₅₀ of 0.0065 μM for anti-proliferative inhibition [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].

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1. In Balb nu/nu mice bearing NCI-H2122 (K-ras-mutated) human lung carcinoma xenografts, RO4987655 was administered at doses of 1.0, 2.5, 5.0 mg/kg. [(¹⁸)F] FDG-PET imaging was used to detect tumor glucose metabolism from day 0 (baseline) to day 9 after treatment: modest decreases in tumor [(¹⁸)F] FDG uptake were observed as early as 2 hours post-treatment; the greatest decrease in [(¹⁸)F] FDG uptake occurred on day 1, followed by a rebound in [(¹⁸)F] FDG uptake on day 3, which was parallel to the decrease in tumor volume. Reverse phase protein arrays (RPPA) analysis of tumor tissues showed that on day 1 after RO4987655 treatment, the expression levels of pERK1/2, pMKK4 and pmTOR were down-regulated, while on day 3, the expression levels of pMEK1/2, pMEK2, pC-RAF and pAKT were significantly up-regulated, indicating the reactivation of MAPK pathway and activation of compensatory PI3K pathway. Semi-quantitative fluorescent immunohistochemistry (fIHC) analysis of tumor tissues found no statistically significant correlation between the expression levels of GLUT1 and hexokinase 1 and the changes in [(¹⁸)F] FDG uptake [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].

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1. Peripheral blood mononuclear cells (PBMCs) assay for pharmacodynamic evaluation (reference [1]): PBMCs were isolated from healthy volunteers who received single oral doses of RO4987655 (0.5, 1, 2, 3, 4 mg). The PBMCs were stimulated ex vivo with PMA, and the changes in phosphorylated extracellular signal-regulated kinase (pERK) levels were detected to evaluate the pharmacodynamic effect of RO4987655. The results showed that pERK inhibition was exposure-dependent, with more than 80% inhibition at higher doses, and the pharmacokinetic-pharmacodynamic relationship was characterized by an inhibitory Eₘₐₓ model (Eₘₐₓ ≈ 100%; IC₅₀ = 40.6 ng/mL) using nonlinear mixed-effect modeling [1]
2. NCI-H2122 cell proliferation and signaling pathway assay (reference [2]): NCI-H2122 cells were seeded in culture plates and cultured to the logarithmic growth phase. For concentration-dependent experiments, cells were treated with RO4987655 at concentrations of 0, 0.01, 0.03, 0.1, 0.3, 1 μM for 2 hours; for time-dependent experiments, cells were treated with 0.1 μM RO4987655 for 0, 6 hours, day 1, day 2, day 3. After treatment, cell lysates were prepared, and immunoblotting was performed using antibodies against phosphorylated and total ERK1/2, MEK1/2, AKT, EGFR, MKK4, Cyclin D1 and Actin (loading control) to detect the phosphorylation levels of related proteins. For anti-proliferative activity assay, NCI-H2122 cells were treated with serial concentrations of RO4987655, and cell proliferation was detected by appropriate methods (e.g., CCK-8, MTT) after a certain culture period; the IC₅₀ value for anti-proliferative inhibition was calculated as 0.0065 μM [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].

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NCI-H2122 tumor xenograft model in Balb nu/nu mice (reference [2]): NCI-H2122 human lung carcinoma cells were subcutaneously inoculated into Balb nu/nu mice to establish tumor xenograft models. When the tumors reached an appropriate volume, RO4987655 was administered to the mice at doses of 1.0, 2.5, 5.0 mg/kg (the specific administration route and frequency were not clearly described in the literature). [(¹⁸)F] FDG-PET imaging (microPET Focus 120) was performed on day 0 (baseline), 2 hours, day 1, day 3, day 9 after the first drug administration to detect the [(¹⁸)F] FDG uptake in tumors (expressed as % ID/gr, injected dose per gram tissue). Tumor volume was measured dynamically during the treatment period. After the experiment, the mice were euthanized, and tumor tissues were collected for semi-quantitative fluorescent immunohistochemistry (fIHC) to detect the expression levels of GLUT1 and hexokinase 1, and reverse phase protein arrays (RPPA) to analyze the phosphorylation levels of MAPK/PI3K pathway components [2]

Objective: CH4987655 (RO4987655) is a small molecule MEK inhibitor with high oral activity and selectivity. It can effectively inhibit the activation of the mitogen-activated protein kinase signaling pathway and tumor cell growth, with an IC50 of 5.2 nmol/L for inhibiting MEK1/2 in vitro. Oral administration of CH4987655 alone can completely regress tumors in xenograft tumor models. [1]
Experimental design: All 40 subjects received a single oral dose, followed by pharmacokinetic, pharmacodynamic and safety/tolerability assessments over 72 hours. Pharmacodynamic assessment was performed by detecting changes in phosphorylated extracellular signal-regulated kinase (pERK) levels in PMA-stimulated peripheral blood mononuclear cells (a substitute tissue). [1]
Results: Doses of 0.5, 1, 2, 3 and 4 mg were safe and well-tolerated. No clinically significant safety events were observed. A total of 26 adverse events were reported (n = 15): 21 were mild, 5 were moderate, and none were severe. One subject experienced a moderate adverse event (autonomic nervous system imbalance) in the 1 mg dose group, and three subjects experienced moderate adverse events (diarrhea, abdominal pain, autonomic nervous system disturbance, and acne) in the 4 mg dose group. CH4987655 is rapidly absorbed, reaching peak concentration in approximately 1 hour. Drug exposure is dose-dependent across the 0.5 to 4 mg dose range. Drug distribution is biphasic, with a terminal half-life of approximately 25 hours. Inter-subject variability was low, with Cmax ranging from 9% to 23% and area under the curve (AUC) from 14% to 25%. pERK inhibition was dose-dependent, with inhibition exceeding 80% at high doses. The pharmacokinetic-pharmacodynamic relationship was characterized by a nonlinear mixed-effects model and an inhibitory E(max) model (E(max) approximately 100%; IC(50) 40.6 ng/mL). [1] Conclusion: A single dose can achieve a significant pERK inhibitory effect, and the drug is safe and well-tolerated in healthy volunteers.

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In healthy volunteers, single oral administration of 0.5, 1, 2, 3, 4 mg of RO4987655 (CH4987655) showed rapid absorption with a peak time (tₘₐₓ) of approximately 1 hour; exposure (Cₘₐₓ and AUC) was dose-proportional in the range of 0.5 to 4 mg; the drug exhibited biphasic distribution with a terminal half-life (t₁/₂) of approximately 25 hours; and low inter-individual variability, with Cₘₐₓ ranging from 9% to 23% and area under the curve (AUC) ranging from 14% to 25%[1]

1. In healthy volunteers, all doses of 0.5, 1, 2, 3, and 4 mg of RO4987655 (CH4987655) were safe and well-tolerated after a single oral dose, and no clinically significant safety events were observed. A total of 26 adverse events were reported in 15 subjects, of which 21 were mild adverse events, 5 were moderate adverse events, and no severe adverse events occurred. Moderate adverse events included one subject who received a 1 mg dose experiencing autonomic nervous system imbalance, and three subjects who received a 4 mg dose experiencing diarrhea, abdominal pain, autonomic nervous system disorder, and acne [1].

[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 clinical trials for tumor treatment research. The MEK inhibitor RO4987655 is an orally active small molecule that targets mitogen-activated protein kinase 1 (MAP2K1 or MEK1) and has potential anti-tumor activity. The MEK inhibitor RO4987655 binds to MEK and inhibits its activity, which may lead to the inhibition of MEK-dependent cell signaling and tumor cell proliferation. MEK is a bispecific threonine/tyrosine kinase and a key component of the RAS/RAF/MEK/ERK signaling pathway, which regulates cell growth; constitutive activation of this pathway is associated with various cancers. Mitogen-activated protein kinase (MEK, also known as MAPK2 or MAPKK) is a key molecule in the Ras/MAPK (mitogen-activated protein kinase) pathway, and its inhibitors have shown good efficacy in clinical trials against B-raf mutations and certain RAS (rat sarcoma)-activated tumors. This study aimed to investigate the efficacy of a novel allosteric MEK inhibitor, RO4987655, in a K-ras-mutant human tumor xenograft model using [(18)F]FDG-PET imaging and proteomics. Methods: [(18)F]FDG uptake in human lung cancer xenografts was studied using a microPET Focus 120 (CTI Concorde Microsystems, Knoxville, TN, USA) from day 0 to day 9 after RO4987655 treatment. Semi-quantitative immunofluorescence (fIHC) was used to detect the expression levels of GLUT1 and hexokinase 1. The in vivo effects of RO4987655 on MAPK/PI3K pathway components were assessed using reverse-phase protein microarray (RPPA). Results: We observed a slight decrease in tumor uptake of [(18)F]FDG 2 hours after MEK inhibition by RO4987655. The most significant decrease in [(18)F]FDG uptake occurred on day 1, followed by a rebound on day 3, coinciding with tumor volume reduction. Molecular analysis of the tumor using fIHC did not reveal a statistically significant correlation between the expression of GLUT1 and hexokinase 1 and the changes in [(18)F]FDG. RPPA signaling pathway response analysis showed that pERK1/2, pMKK4, and pmTOR expression was downregulated on day 1 after RO4987655 treatment, while pMEK1/2, pMEK2, pC-RAF, and pAKT expression was significantly upregulated on day 3. The upregulation of these markers was interpreted as a sign of MAPK pathway reactivation and compensatory PI3K pathway activation, which could also explain the rebound in [(18)F]FDG uptake after MEK inhibition with RO4987655 in a K-ras mutant human tumor xenograft model. Conclusion: We are the first to use a combination of [(18)F]FDG-PET imaging and molecular proteomics to conduct a preclinical evaluation of a novel MEK inhibitor, RO4987655. These results support the use of preclinical [(18)F]FDG-PET imaging for early, non-invasive monitoring of the effects of MEK and other Ras/MAPK signaling pathway inhibitors, which should facilitate wider clinical implementation of [(18)F]FDG-PET to optimize its clinical application. [2]

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1. RO4987655 (CH4987655) is a small molecule allosteric MEK inhibitor with high oral activity and selectivity; oral monotherapy with RO4987655 can completely regress tumors in xenograft models[1]
2. In human tumor xenograft models with K-ras mutations, the rebound of [(¹⁸)F]FDG uptake after RO4987655 treatment was explained as being caused by MAPK pathway reactivation and compensatory PI3K pathway activation[2]
3. [(¹⁸)F]FDG-PET imaging combined with molecular proteomics can be used for preclinical assessment of the efficacy of RO4987655. This method supports the application of [(¹⁸)F]FDG-PET in clinical practice to non-invasively monitor the effects of MEK inhibitors and optimize their clinical application[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.)