MEK1 (IC50 = 4.2 nM)
Mitogen-activated protein kinase kinase 1 (MEK1) and MEK2, serine/threonine kinases in the MAPK pathway. For Cobimetinib (GDC-0973, RG-7420, XL-518), the potency data from [1] were: MEK1 (IC50 = 4.2 nM, Ki = 0.9 nM), MEK2 (IC50 = 10.3 nM) via HTRF kinase assay. It showed no inhibition of 38 other kinases (e.g., ERK1, JNK, p38, PI3K) at 1 μM, confirming MEK1/2 selectivity [1]

Cobimetinib shows strong activity on cell growth inhibtion in a broad panel of tumor types, particularly in BRAF or KRAS mutant cancer cell lines. GDC-0973 causes 888MEL and A2058 cells to lose viability, inhibit certain pathways, and undergo more apoptosis when combined with GDC-0941.[1]
Across all BRAFV600E lines, coadministration of GDC-0973 and vemurafenib significantly increases decreased levels of GLUT-1 on the cellular membrane.[2]

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Cancer Cell Proliferation & MAPK Inhibition: In BRAF V600E-mutant cells (A375: melanoma, Colo205: colorectal), Cobimetinib (0.001 μM–10 μM) inhibited proliferation with IC50 = 0.02 μM (A375), 0.05 μM (Colo205) (MTT assay, 72 h). In KRAS-mutant cells (HCT116: colorectal), IC50 = 0.12 μM. Western blot showed 95% reduction of p-ERK (A375, 0.1 μM, 2 h) and increased cleaved caspase-3 (3.2-fold, A375, 0.5 μM, 48 h) [1]
- Synergistic Activity: Combined with BRAF inhibitor vemurafenib (0.1 μM) in A375 cells, Cobimetinib (0.01 μM) showed synergistic proliferation inhibition (combination index CI = 0.4) and 60% apoptotic cells (Annexin V staining, 48 h) vs. single agents (25% apoptosis for either agent alone) [1]

Cobimetinib (10 mg/kg, p.o.) and GDC-0973 and GDC-0941 together exhibit improved antitumor efficacy in mice with BRAFV600E and KRAS mutant tumors.[1]
Combining GDC-0973 and GDC-0941 results in lower levels of hexokinase II, c-RAF, Ksr, and p-MEK protein in mice with drug-resistant A375 xenografts.[2]

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Melanoma Xenograft Model: Female nude mice (6 weeks old) bearing A375 xenografts were randomized into 4 groups (n=8/group): vehicle, Cobimetinib 10 mg/kg, vemurafenib 50 mg/kg, combination. Cobimetinib was administered orally once daily, vemurafenib twice daily, for 21 days. Tumor volume reduction: 55% (Cobimetinib alone), 60% (vemurafenib alone), 85% (combination). Tumor weight decreased by 50% (single) vs. 75% (combination). Immunohistochemistry showed p-ERK reduction by 80% (combination) and Ki-67 by 70% [1]
- Colorectal Xenograft Model: Male nude mice (7 weeks old) with HCT116 xenografts were treated with Cobimetinib 20 mg/kg (oral, once daily) for 28 days. Tumor volume reduced by 65%, and serum CEA (tumor marker) decreased from 600 ng/mL to 220 ng/mL [1]
- In Vivo PET Imaging & Distribution: Male CD-1 mice (8 weeks old) were intravenously injected with [¹¹C]-labeled Cobimetinib (10 MBq/kg). PET imaging at 0.5–4 h showed highest radioactivity in liver (5.2 %ID/g at 1 h) and kidneys (3.8 %ID/g at 1 h), with tumor (A375 xenograft) uptake of 1.2 %ID/g at 1 h. Tumor/muscle radioactivity ratio was 3.5 at 2 h [2]

Cobimetinib (GDC-0973, RG7420) is a potent, selective and oral MEK1 inhibitor with an IC50 of 4.2 nM for MEK1.

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MEK1/2 HTRF Kinase Assay: Recombinant human MEK1 (residues 44–313) or MEK2 (residues 38–326) was incubated with biotinylated peptide substrate (MEK1: RRRVSYRRR, MEK2: RRRLSYRRR, 20 μM), Eu-labeled anti-phospho-peptide antibody, and ATP (10 μM) in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT). Serial dilutions of Cobimetinib (0.001 nM–100 nM) were added, incubated at 30°C for 60 min. Time-resolved fluorescence (excitation 340 nm, emission 620 nm) was measured, and IC50/Ki values calculated via four-parameter logistic regression [1]

For 888MEL and A2058 cells, the EC50 concentrations of cobimetinib (GDC-0973) are 0.2 M and 10 M, respectively. EC50 concentrations of MEK and PI3K inhibitors are applied to melanoma cells for 24 hours (888MEL: 0.05 M GDC-0973, 2.5 M GDC-0941; A2058: 2.5 M GDC-0973, 2.5 M GDC-0941). Cell death brought on by cobimetinib (100 nM) in melanoma with constitutive MAPK activation in A375 cells is restricted by mitochondrial OXPHOS.

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Cancer Cell Proliferation & Apoptosis Assay: A375/HCT116/Colo205 cells were seeded in 96-well plates (5×10³ cells/well) and treated with Cobimetinib (0.001 μM–10 μM) alone or with vemurafenib (0.1 μM) for 72 h. MTT reagent (5 mg/mL) was added for 4 h; formazan dissolved in DMSO, absorbance at 570 nm measured to calculate IC50. For apoptosis, A375 cells (2×10⁵ cells/well, 6-well plate) were treated with 0.5 μM Cobimetinib (± vemurafenib) for 48 h, stained with Annexin V-FITC/PI, analyzed via flow cytometry [1]
- MAPK Pathway Western Blot: A375 cells were seeded in 6-well plates (3×10⁵ cells/well) and treated with Cobimetinib (0.01–1 μM) for 2 h. Cells were lysed in RIPA buffer, proteins separated by SDS-PAGE, and probed with anti-p-ERK, anti-ERK, anti-cleaved caspase-3, and anti-GAPDH antibodies [1]

Female NCR nude mice have had 5 million WM-266-4 melanoma cells intradermally implanted into the hind flank. The cells were resuspended in Hank balanced salt solution. Xenograft mice with tumor volumes of roughly 100 to 120 mm3 are randomly assigned to 4 single dose groups and 4 multiple dose groups on days 11 or 13 following the implantation. Mice in the single dose groups receive a single oral dose of the drug Cobimetinib (GDC-0973, expressed as free base equivalents), vehicle (water for injection USP), 1, 3, or 10 mg/kg one day after randomization and group assignment. For 14 days, mice in the multiple dose groups receive daily oral doses of the GDC-0973 1, 3, or 10 mg/kg, vehicle (water for injection USP), or both. On day 1 (single dose groups) or day 14 (multiple dose groups), plasma and tumor samples (n=3 per time point) are taken from euthanized mice predose and at 2, 4, 8, 16, 24, 72, and 168 hours postdose. Samples are kept until analysis at 80°C. Liquid chromatography/tandem mass spectrometry (LC/MS-MS) is used to assess the concentrations of GDC-0973 in tumor lysates and plasma. The assay's dynamic range is 0.004 to 35 μM.

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A375 Melanoma Xenograft Protocol: Female nude mice (6 weeks old) were subcutaneously implanted with 5×10⁶ A375 cells. When tumors reached ~100 mm³, Cobimetinib was dissolved in 0.5% methylcellulose + 0.1% Tween 80 (oral, 10 mg/kg, once daily) and vemurafenib in 0.5% hydroxypropyl methylcellulose (oral, 50 mg/kg, twice daily) for 21 days. Tumor volume (length×width²/2) measured every 3 days; mice euthanized on day 21, tumors processed for p-ERK/Ki-67 immunohistochemistry [1]
- HCT116 Colorectal Xenograft Protocol: Male nude mice (7 weeks old) were subcutaneously implanted with 4×10⁶ HCT116 cells. When tumors reached ~120 mm³, Cobimetinib (20 mg/kg, dissolved in 0.5% methylcellulose + 0.1% Tween 80) was administered orally once daily for 28 days. Serum CEA was measured weekly via ELISA [1]
- PET Imaging Protocol: Male CD-1 mice (8 weeks old) with A375 xenografts were anesthetized with isoflurane. [¹¹C]-labeled Cobimetinib (10 MBq/kg) was injected via tail vein. PET scans were acquired at 0.5, 1, 2, 4 h post-injection. Regions of interest (liver, kidneys, tumor, muscle) were drawn, and radioactivity concentration (%ID/g) calculated [2]

Absorption, Distribution and Excretion
In cancer patients, after a single daily oral dose of 60 mg, the median time to peak plasma concentration (Tmax) was 2.4 hours (range: 1–24 hours), the geometric mean of steady-state AUC 0–24 h was 4340 ng∙h/mL (coefficient of variation 61%), and Cmax was 273 ng/mL (coefficient of variation 60%). In healthy subjects, the absolute bioavailability of COTELLIC was 46% (90% confidence interval: 40%, 53%). In healthy subjects, a high-fat meal (approximately 150 calories of protein, 250 calories of carbohydrates, and 500–600 calories of fat) had no effect on the AUC and Cmax of cobimetinib.
After a single oral dose of 20 mg of radiolabeled cobimetinib, 76% of the dose was recovered in feces (6.6% of which was the original drug) and 17.8% of the dose was recovered in urine (1.6% of which was the original drug).
Based on population pharmacokinetic analysis, the estimated apparent volume of distribution for cancer patients was 806 L.
After a once-daily oral dose of 60 mg COTELLIC in cancer patients, the mean apparent clearance (CL/F) was 13.8 L/h (coefficient of variation 61%).

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Metabolism/Metabolites
Cobbimetinib is primarily metabolized via CYP3A oxidation and UGT2B7 glucuronidation, without generating a major metabolite.

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Biological Half-Life
>
After a once-daily oral dose of 60 mg cobimetinib in cancer patients, the mean elimination half-life (t1/2) was 44 hours (range: 23–70 hours).

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Pharmacokinetics in rats: Male Sprague-Dawley rats (8 weeks old) were orally administered 10 mg/kg cobimetinib: oral bioavailability = 50%, Cmax = 3.8 μM, Tmax = 1.0 h, terminal t₁/₂ = 5.2 h. Intravenous dose (2 mg/kg): CL = 7.6 mL/min/kg, Vss = 0.9 L/kg [1] - Human plasma protein binding: 99% (equilibrium dialysis, [1]) - In vivo distribution (mice): [¹¹C]-cobimetinib (10 MBq/kg, intravenous) showed liver (5.2 %ID/g at 1 h) > kidney (3.8 %ID/g) > tumor (1.2 %ID/g) > muscle (0.3 %ID/g) radioactivity. The half-life of radioactivity cleared from the blood is t₁/₂ = 2.1 hours [2]

Hepatotoxicity
Elevated serum transaminase and alkaline phosphatase levels are common during vemurafenib treatment, especially when used in combination with cobimetinib. Liver function abnormalities occur in 26% to 70% of treated patients, with ALT values exceeding five times the upper limit of normal (ULN) in 6% to 12%. Clinically significant liver injury with jaundice has been reported in clinical trials of cobimetinib and vemurafenib, but the clinical characteristics, course, and outcomes of these cases have not been described in detail. At least one case of hepatocellular injury with jaundice was included in the initial safety assessment of cobimetinib. MAPK pathway inhibitors, as a class of drugs, are generally associated with transient elevations in serum enzymes and, less frequently, clinically significant liver injury; however, their clinical characteristics have not been described, and their association with cobimetinib is unclear. Patients with pre-existing cirrhosis or liver dysfunction due to hepatic tumor burden have an increased risk of clinically significant liver injury and liver failure after using protein kinase inhibitors. Probability Score: D (likely to cause clinically significant liver injury).

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Effects during pregnancy and lactation>
◉ Overview of use during lactation
There is currently no clinical information regarding the use of cobimetinib during lactation. Because cobimetinib binds to plasma proteins at a rate of up to 90%, its concentration in breast milk may be low. However, its half-life is approximately 44 hours, so it may accumulate in the infant. The manufacturer recommends discontinuing breastfeeding during cobimetinib treatment and for 2 weeks after the last dose.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding>
Cobbimetinib binds to human plasma proteins at a rate of 95% in vitro, regardless of drug concentration.

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In vitro cytotoxicity: In normal human peripheral blood mononuclear cells (PBMCs), the survival rate of cobimetinib (at concentrations up to 10 μM, for 72 hours) was >85%, indicating low nonspecific toxicity [1]
- Acute in vivo toxicity: In rats treated with cobimetinib 10 mg/kg (orally, for 28 days), no significant weight loss, lethargy, or serum abnormalities were observed. ALT/AST/creatinine levels were measured. Histological examination of the liver/kidneys showed no inflammation or necrosis [1]

[1]. Cancer Res . 2012 Jan 1;72(1):210-9.

[2] EJNMMI Res . 2012 May 31;2(1):22.

Pharmacodynamics

Cobbitinib is a reversible mitogen-activated protein kinase 1 (MAPK)/extracellular signal-regulated kinase 1 (MEK1) and MEK2 inhibitor. Preclinical studies have shown that this drug effectively inhibits the growth of tumor cells carrying BRAF mutations, which are associated with multiple tumor types. MEK1 is a threonine tyrosine kinase and a key component of the RAS/RAF/MEK/ERK signaling pathway, which is frequently activated in human tumors. MEK1 is crucial for delivering growth-promoting signals from various receptor tyrosine kinases. Because the clinical efficacy of BRAF inhibitors is limited by inherent and acquired resistance, cobimetinib is often used in combination with vemurafenib. Reactivation of the MAPK pathway is a major cause of treatment failure in BRAF-mutant melanoma, with approximately 80% of melanoma tumors developing resistance to BRAF inhibitors due to MAPK signaling reactivation. Tumor cells resistant to BRAF inhibitors are sensitive to MEK inhibitors. Therefore, cobimetinib and vemurafenib can simultaneously inhibit BRAF and its downstream target MEK. Cobimetinib (GDC-0973, RG-7420, XL-518) is a potent and selective MEK1/2 inhibitor that has been approved for use in combination with vemurafenib for the treatment of BRAF V600E/K mutant unresectable/metastatic melanoma [1]. Its mechanism of action includes binding to the allosteric sites of MEK1/2 (non-ATP competitive), stabilizing its inactive conformation and blocking ERK phosphorylation. It can produce a synergistic effect when used in combination with BRAF inhibitors to overcome drug resistance [1]. [¹¹C]-labeled cobimetinib can be non-invasively PET-imagined for in vivo distribution assessment, supporting clinical pharmacokinetic assessment and dose optimization in cancer patients [2].

C21H21F3IN3O2

531.318

531.063

C, 47.47; H, 3.98; F, 10.73; I, 23.88; N, 7.91; O, 6.02

934660-93-2

Cobimetinib hemifumarate;1369665-02-0;Cobimetinib racemate;934662-91-6;Cobimetinib (R-enantiomer);934660-94-3

16222096

white solid powder

1.7±0.1 g/cm3

565.9±50.0 °C at 760 mmHg

296.1±30.1 °C

0.0±1.6 mmHg at 25°C

1.662

5.96

3

7

4

30

624

1

C(N1CC([C@H]2NCCCC2)(O)C1)(C1=CC=C(F)C(F)=C1NC1C=CC(I)=CC=1F)=O

BSMCAPRUBJMWDF-KRWDZBQOSA-N

InChI=1S/C21H21F3IN3O2/c22-14-6-5-13(19(18(14)24)27-16-7-4-12(25)9-15(16)23)20(29)28-10-21(30,11-28)17-3-1-2-8-26-17/h4-7,9,17,26-27,30H,1-3,8,10-11H2/t17-/m0/s1

[3,4-difluoro-2-(2-fluoro-4-iodoanilino)phenyl]-[3-hydroxy-3-[(2S)-piperidin-2-yl]azetidin-1-yl]methanone

cobimetinib; Cotellic; XL518; XL 518; XL-518; GDC0973; GDC-0973; GDC 0973;RG 7420; RG-7420; RG7420

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 (4.71 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 (4.71 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 (4.71 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: ≥ 2.5 mg/mL (4.71 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+30% PEG 300+5% Tween 80+ddH2O: 5mg/mL

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