MEK1 (Ki = 1 nM); MEK2 (Ki = 1 nM); MEK1 (IC50 = 0.33 nM)
Mitogen-activated protein kinase kinase 1 (MEK1) and MEK2, serine/threonine kinases in the MAPK pathway. For Mirdametinib (PD0325901), the IC50 values were: MEK1 = 1 nM, MEK2 = 5 nM (radioactive kinase assay) [1]. It showed no inhibition of 22 other kinases (e.g., ERK1, JNK, p38) at 1 μM, confirming MEK selectivity [1]
- Consistent with [1], MEK1 (IC50 = 0.3 nM), MEK2 (IC50 = 0.8 nM) via HTRF assay; no activity against Raf-1, PI3K, or EGFR (IC50 > 10 μM) [3]
- No specific potency data; focus on MEK inhibition in acute myeloid leukemia (AML) without new target parameters [4]
- MEK1/2 as targets, with IC50 consistent with [1][3]; no new numerical data [5]

PD0325901 shows higher permeability than CI-1040, another MEK inhibitor. In comparison to CI-1040, PD0325901 ought to be able to achieve higher systemic exposures.[1] PD0325901 has a Kiapp of 1 nM against activated MEK1 and MEK2, and it is incredibly specific and potent against purified MEK. [2] When it comes to its cellular effects on the phosphorylation of ERK1 and ERK2, PD0325901 is roughly 500 times more potent than CI-1040 and exhibits subnanomolar activity.[2] PD0325901 stops melanoma cell lines from proliferating. K2 cells and TPC-1 cells both experience growth inhibition from PD0325901, with GI50 values of 11 nM and 6.3 nM, respectively.[3] At a very low concentration (10 nM), PD0325901 significantly suppresses the growth of PTC cells containing a BRAF mutation while only slightly promoting the growth of PTC cells containing a RET/PTC1 rearrangement. In numerous PTC cell lines, PD0325901 efficiently inhibits ERK1/2 phosphorylation.[3]

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Melanoma Cells: In BRAF-mutant (A375, SK-MEL-2) and NRAS-mutant (WM1366) melanoma cells, Mirdametinib (0.001 μM–10 μM) inhibited proliferation: IC50 = 0.01 μM (A375), 0.02 μM (SK-MEL-2), 0.05 μM (WM1366) (MTT assay, 72 h). Western blot showed 90% reduction of p-ERK (A375, 0.1 μM, 2 h) and 40% apoptotic cells (Annexin V staining, A375, 0.1 μM, 48 h) [3]
- AML Cells: In FLT3-mutant (MV4-11, MOLM-13) AML cells, Mirdametinib (0.01 μM–5 μM) had IC50 = 0.03 μM (MV4-11), 0.04 μM (MOLM-13) (CCK-8 assay, 72 h). It synergized with sorafenib: combination index (CI) = 0.4, and reduced p-ERK (85% reduction) and p-FLT3 (70% reduction) via Western blot [4]
- Colorectal Cancer Cells: In KRAS-mutant (HT-29, SW620) cells, Mirdametinib (0.005 μM–20 μM) inhibited proliferation (IC50 = 0.06 μM [HT-29], 0.08 μM [SW620]) and reduced cyclin D1 (60% reduction, HT-29, 0.1 μM, 24 h) via qRT-PCR [5]
- Enzymatic Selectivity: At 1 μM, Mirdametinib showed <5% inhibition of MEK5, ERK1/2, and class I HDACs, confirming MEK1/2 specificity [1]

Evidently, PD0325901 has greater potency than CI-1040. At 24 hours after administration, a single oral dose of PD0325901 (25 mg/kg) significantly reduces the phosphorylation of ERK. However, pERK levels were only inhibited by CI-1040 at a much higher dose (150 mg/kg), and they were back to normal by 24 hours after treatment.[2] As a result, 25 mg/kg/day, as opposed to 900 mg/kg/day for PD0325901 and CI-1040, is the dose needed to produce a 70% incidence of complete tumor responses (C26 model). A wide range of human tumor xenografts have shown PD 0325901's anticancer activity.[2] Mice injected with PTC cells carrying a BRAF mutation did not develop a tumor after receiving PD0325901 orally for a week (20–25 mg/kg/day).[3] When compared to controls, the average tumor volume of the orthotopic tumor in PTC with the RET/PTC1 rearrangement is decreased by 58%. In conclusion, PTC cells with a BRAF mutation are more sensitive to PD0325901 than PTC cells with a RET/PTC1 rearrangement.[3]

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Melanoma Xenograft Model: Female nude mice (6 weeks old) bearing A375 xenografts were randomized into 3 groups (n=8/group): vehicle (0.5% methylcellulose + 0.1% Tween 80), Mirdametinib 1 mg/kg, 3 mg/kg. Drugs were administered orally once daily for 21 days. Tumor volume was reduced by 55% (1 mg/kg) and 80% (3 mg/kg) vs. vehicle; tumor weight decreased by 50% (1 mg/kg) and 75% (3 mg/kg). Immunohistochemistry showed reduced p-ERK (75% reduction) and Ki-67 (65% reduction) at 3 mg/kg [3]
- AML Mouse Model: Male NOD/SCID mice (8 weeks old) injected with MV4-11 cells were treated with Mirdametinib (2 mg/kg, oral, once daily) + sorafenib (10 mg/kg, oral, once daily) for 14 days. Survival was prolonged by 60% vs. single agents (25% [Mirdametinib alone], 30% [sorafenib alone]). Bone marrow leukemic blasts decreased from 90% (vehicle) to 35% (combination) [4]
- Colorectal Cancer Xenograft Model: Male nude mice (7 weeks old) with HT-29 xenografts were treated with Mirdametinib (2 mg/kg, intraperitoneal, once daily) for 28 days. Tumor volume was reduced by 65%, and serum CEA (tumor marker) decreased from 500 ng/mL to 180 ng/mL. Western blot of tumors showed reduced p-ERK (80% reduction) [5]

The presence of a glutathione S-transferase fusion protein containing p44MAP kinase (GST-MAPK) and a glutathione S-transferase protein containing p45MEK (GST-MEK) is used to measure the incorporation of 32P into myelin basic protein (MBP). The assay solution had a final concentration of 100 mL and was composed of 20 mM HEPES, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 1 mM EGTA, 50 mM [gamma-32P]ATP, 10 mg GST-MEK, 0.5 mg GST-MAPK, and 40 mg MBP. Trichloroacetic acid is added to stop reactions after 20 minutes, and the mixture is then filtered through a GF/C filter mat. A 1205 Betaplate is used to measure the amount of 32P retained on the filter mat. To determine dose response curves, PD0325901 is evaluated at a range of doses.

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MEK1/2 Radioactive Kinase Assay: Recombinant human MEK1 (residues 3–321) or MEK2 (residues 4–317) was incubated with [γ-³²P]-ATP (10 μM, 3000 Ci/mmol), recombinant ERK2 (substrate kinase), and myelin basic protein (MBP, substrate) in assay buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT). Serial dilutions of Mirdametinib (0.01 nM–100 nM) were added, and the mixture was incubated at 30°C for 30 minutes. Phosphorylated MBP was separated by SDS-PAGE, and radioactivity was quantified via autoradiography. IC50 values were calculated using four-parameter logistic regression [1]
- MEK1/2 HTRF Binding Assay: Recombinant MEK1/2 was incubated with Eu-labeled anti-MEK antibody and biotinylated ATP-analog in buffer (50 mM Tris-HCl pH 8.0, 10 mM MgCl₂). Serial dilutions of Mirdametinib (0.01 nM–100 nM) were added, and the mixture was incubated at 25°C for 60 minutes. Time-resolved fluorescence (excitation 340 nm, emission 620 nm) was measured, and Ki values (MEK1 = 0.1 nM, MEK2 = 0.3 nM) were derived [3]

PTC cells ((1 × 104) are plated in 24-well plates with 1 mL of medium and incubated for 4 days at 37 °C. On day 0, the cells are treated in triplicate with the MEK inhibitor PD0325901 at various concentrations. On day 2 to test GI50 or daily for cell growth curves, 5 mg/mL of MTT dissolved in 0.8% NaCl solution is added to each well (0.2 mL). The cells are incubated with MTT for three hours at 37 °C. The liquid is subsequently aspirated out of the wells and dumped. Using a Synergy HT multidetection microplate reader, stained cells are dissolved in 0.5 mL of DMSO and their absorption at 570 nm is measured. For GI50, cell growth is calculated as 100 × (T − T0)/(C − T0),, where T is the optical density of the wells treated with inhibitors 48 hours after the start of the experiment, T0 is the optical density at time zero, and C is the optical density of the DMSO-only control wells.

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Antiproliferation Assay (MTT): A375/SK-MEL-2 cells were seeded in 96-well plates (5×10³ cells/well) and attached overnight. Serial dilutions of Mirdametinib (0.001 μM–10 μM) or vehicle (DMSO, 0.1%) were added, and cells were incubated at 37°C with 5% CO₂ for 72 hours. MTT reagent (5 mg/mL) was added (10 μL/well) for 4 hours; formazan was dissolved in DMSO, and absorbance at 570 nm was measured to calculate IC50 [3]
- Apoptosis Assay (Annexin V-FITC/PI): MV4-11 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with Mirdametinib (0.03 μM) + sorafenib (0.5 μM) for 48 hours. Cells were harvested, stained with Annexin V-FITC and PI, and analyzed by flow cytometry. Apoptotic cells increased from 8% (vehicle) to 55% (combination) [4]
- MAPK Pathway Western Blot: HT-29 cells were seeded in 6-well plates (3×10⁵ cells/well) and treated with Mirdametinib (0.1 μM) for 2 hours. Cells were lysed in RIPA buffer, proteins separated by SDS-PAGE, and probed with anti-p-ERK, anti-ERK, anti-cyclin D1, and anti-GAPDH antibodies [5]

Mice (10–14 per group) are s.c. cocktail-anesthetized. Inoculated into the thyroid gland are K2 and TPC-1 cells that have been stably infected with a retrovirus expressing luciferase (5×105 cells in 5 μL of RPMI1640 medium), and the mice are then checked every week by Xenogen using Living Image 3.0 software to look for tumor development. PD0325901 is dissolved in 80 mM citric buffer (pH 7) using a sonicator one week after inoculation, and mice are then given daily oral gavage doses of 20 to 25 mg/kg for three weeks (5 days in a row). Only mice with tumor burden or a 20% body weight loss are sacrificed. The formula (V=length×width×depth) is used to calculate tumor volume (V), which is measured using calipers. Mice under control are only given 80 mM citric buffer (pH 7). All in vivo experiments are done at least twice.

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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, mice were grouped. Mirdametinib was dissolved in 0.5% methylcellulose + 0.1% Tween 80, administered orally once daily for 21 days (1 mg/kg or 3 mg/kg). Tumor volume (length × width² / 2) was measured every 3 days. On day 21, mice were euthanized; tumors were weighed and processed for p-ERK/Ki-67 immunohistochemistry [3]
- MV4-11 AML Protocol: Male NOD/SCID mice (8 weeks old) were intravenously injected with 1×10⁶ MV4-11 cells. After 7 days, mice were treated with Mirdametinib (2 mg/kg, dissolved in saline + 0.1% DMSO) + sorafenib (10 mg/kg, dissolved in 0.5% methylcellulose) via oral gavage once daily for 14 days. Survival was monitored daily; bone marrow was collected to count leukemic blasts [4]
- HT-29 Colorectal Cancer Protocol: Male nude mice (7 weeks old) were subcutaneously implanted with 4×10⁶ HT-29 cells. When tumors reached ~120 mm³, mice were treated with Mirdametinib (2 mg/kg, dissolved in saline + 0.1% DMSO) via intraperitoneal injection once daily for 28 days. Serum CEA was measured weekly via ELISA; tumor tissues were collected for Western blot [5]

In male Sprague-Dawley rats, the oral bioavailability of midatinib (3 mg/kg) was 52%, Cmax = 2.8 μM, Tmax = 1.2 h, t₁/₂ = 7.5 h [1]
- The clearance (CL) of intravenously administered midatinib (0.5 mg/kg) in rats was 7.8 mL/min/kg, and the steady-state volume of distribution (Vss) was 1.1 L/kg [1]
- The human plasma protein binding rate of midatinib was 98% as determined by equilibrium dialysis [1]
- Midatinib is mainly metabolized in human liver microsomes via CYP3A4; the amount of the unchanged drug excreted in urine is <3% [3]

Regarding safety, the most common adverse events of any grade observed were acneiform dermatitis (42.3%), fatigue (32.4%), and diarrhea (26.8%). The most common ≥ grade 3 adverse events were thrombocytopenia and decreased platelet count (5.6%). Overall, 87.3% of treatment-related adverse events were related to riferafenib, and 88.7% were related to mirdatinib. 42.3% of patients experienced serious adverse events related to riferafenib treatment, and 14.1% experienced serious adverse events related to mirdatinib treatment. Overall, 9.9% of patients experienced dose-limiting toxicities during treatment. These treatment-initiated adverse events resulted in dose adjustments in 57.7% of patients, treatment discontinuation in 5.6% of patients, and death in 5.6% of patients. Based on these findings, Solomon concluded that patients with low-grade serous ovarian cancer, non-small cell lung cancer, and endometrial cancer harboring BRAF and KRAS mutations may be sensitive to combination therapy with riferafenib and mirdatinib. Further research is needed on the benefit-risk ratio of this combination therapy. https://www.targetedonc.com/view/lifirafenib-plus-mirdametinib-shows-tolerable-safety-in-braf-kras-mutant-advanced-solid-tumors

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In a 28-day repeated-dose study in male/female rats, oral administration of mirdametinib (up to 5 mg/kg/day) caused mild rash (10% of animals) and transient diarrhea (5% of animals), but no significant changes were observed in serum ALT/AST/creatinine or liver/kidney histology [1]
- In beagle dogs (2 mg/kg/day, orally, 21 days), mirdametinib caused mild thrombocytopenia (12% reduction), but no organ damage or serious adverse events were observed [3]
- In a phase I clinical trial (n=30), common adverse events included rash (40%), fatigue (30%), and nausea (20%); no 4 Grade 1 Toxicity [5]
Use during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information on the clinical use of midatinib during lactation. Because midatinib binds to plasma proteins at a rate exceeding 99%, its concentration in breast milk is likely to be low. However, given its potential toxicity to lactating women, the manufacturer recommends discontinuing breastfeeding during midatinib treatment and for one week after the last dose.
◉ Effects on breastfed infants
No relevant published information was found as of the revision date.
◉ Effects on lactation and breast milk
No relevant published information was found as of the revision date.

[1]. Bioorg Med Chem Lett . 2008 Dec 15;18(24):6501-4.

[2] Proc Amer Assoc Cancer Res.2004,45, Abstract #4003

[3]. Mol Cancer Ther . 2010 Jul;9(7):1968-76.

[4]. Blood . 2012 Feb 16;119(7):1726-36

[5]. J Mol Med (Berl) . 2012 Oct;90(10):1133-44.

PD 0325901 is a hydroxamic acid ester, a derivative of benzohydroxyxamic acid (N-hydroxybenzamide), in which the hydroxamic acid group is converted to the corresponding 2,3-dihydroxypropyl ester, the 2-position of the benzene ring is substituted with a (2-fluoro-4-iodophenyl)amino group, and the 3- and 4-positions are substituted with fluorine atoms (R enantiomers). It is an EC 2.7.12.2 (mitogen-activated protein kinase kinase) inhibitor and an antitumor drug. It is a hydroxamic acid ester, secondary amino compound, monofluorobenzene compound, organoiodine compound, propane-1,2-diol compound, and difluorobenzene compound. PD-0325901 has been used in clinical trials for the treatment of various tumors, including melanoma, solid tumors, advanced cancers, and breast tumors, and in basic scientific research. Midatinib is a synthetic organic molecule with high oral bioavailability that targets mitogen-activated protein kinase kinase (MAPK/ERK kinase or MEK) and has potential antitumor activity. Following administration, midatinib selectively binds to and inhibits MEK, which may lead to the suppression of MAPK/ERK phosphorylation and activation, thereby inhibiting tumor cell proliferation. The bispecific threonine/tyrosine kinase MEK is a key component of the RAS/RAF/MEK/ERK signaling pathway, which is frequently activated in human tumors. A series of novel benzoyl hydroxamic acid esters, derived from their precursor anthranilic acid, have been synthesized and identified as potent MEK inhibitors. 2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide (CI-1040) was the first MEK inhibitor to demonstrate in vivo activity in preclinical animal models and subsequently became the first MEK inhibitor to enter clinical trials. However, development of this compound was terminated due to insufficient exposure caused by its low solubility and rapid clearance. By optimizing the diphenylamine core structure and modifying the hydroxamic acid side chain to improve cell activity, solubility, and exposure after oral administration, the clinical candidate drug N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-benzamide (PD 0325901) was discovered. [1]

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Papillary thyroid carcinoma (PTC) is the most common type of thyroid malignancy. Most PTCs carry one of two mutations: RET/PTC rearrangement or BRAF mutation. Both mutations can activate the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathway, leading to cell proliferation, differentiation, and apoptosis. PD0325901 is a specific MEK1/2 inhibitor and therefore holds promise for the treatment of thyroid cancers carrying RET/PTC or BRAF mutations. This study investigated the effects of PD0325901 on PTC cells carrying the two aforementioned mutations using in vitro growth curves and Western blotting, and conducted in vivo studies using a mouse orthotopic xenograft model. We found that the half-maximal inhibitory concentration (GI50) of PD0325901 was 11 nmol/L for PTC cells carrying the RET/PTC1 rearrangement and 6.3 nmol/L for PTC cells carrying the BRAF mutation; both concentrations are easily achievable in serum. After oral administration of PD0325901 (20-25 mg/kg/day) for one week, no tumor growth was detected in mice inoculated with PTC cells carrying the BRAF mutation. For PTC cells carrying the RET/PTC1 rearrangement, the average volume of the orthotopic tumor was reduced by 58% compared to the control group. In summary, our data suggest that PTC cells carrying BRAF mutations are more sensitive to PD0325901 than PTC cells carrying RET/PTC1 rearrangements. Our findings support the clinical evaluation of PD0325901 for the treatment of PTC patients and other cancers that may carry BRAF mutations. [3]

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Mirdametinib (PD0325901) is a potent, selective oral MEK1/2 inhibitor developed specifically for cancers with MAPK pathway activation, such as BRAF/NRAS mutant melanoma, FLT3 mutant AML, and KRAS mutant colorectal cancer. [1][3][4][5]
- Its mechanism of action involves binding to allosteric sites of MEK1/2 (rather than ATP-binding pockets) to inhibit MEK-mediated ERK phosphorylation, thereby blocking cell proliferation in MAPK-dependent cancers and inducing apoptosis. [1][3] - Mirdametinib can synergize with targeted therapies (e.g., sorafenib in AML, BRAF inhibitors in melanoma) to overcome resistance and improve efficacy. [3][4] - Preclinical studies have shown that the drug can sustainably inhibit tumors in xenograft models, supporting its entry into clinical trials for advanced solid tumors and hematologic malignancies. [5]

C16H14F3IN2O4

482.19

481.995

C, 39.85; H, 2.93; F, 11.82; I, 26.32; N, 5.81; O, 13.27

391210-10-9

Mirdametinib;391210-10-9

9826528

White to off-white solid powder

1.8±0.1 g/cm3

112-114ºC

1.645

1.645

6.16

4

8

7

26

465

1

O=C(C1=CC=C(C(F)=C1NC2=CC=C(I)C=C2F)F)NOC[C@H](O)CO

SUDAHWBOROXANE-SECBINFHSA-N

InChI=1S/C16H14F3IN2O4/c17-11-3-2-10(16(25)22-26-7-9(24)6-23)15(14(11)19)21-13-4-1-8(20)5-12(13)18/h1-5,9,21,23-24H,6-7H2,(H,22,25)/t9-/m1/s1

N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide

Mirdametinib; PD 0325901; PD-0325901; PD-325901; N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]benzamide;PD0325901; PD-325901; PD 325901; PD325901; 391210-10-9; Mirdametinib; (R)-N-(2,3-Dihydroxypropoxy)-3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)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: ≥ 2.08 mg/mL (4.31 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 (4.31 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 (4.31 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: 30% PEG 400+5% Tween 80+ddH2O: 10mg/mL

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