MEK1 (Ki = 1 nM); MEK2 (Ki = 1 nM); MEK1 (IC50 = 0.33 nM)

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]

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]

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.

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.

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.

In terms of safety, the most commons any-grade AEs observed were dermatitis acneiform (42.3%), fatigue (32.4%), and diarrhea (26.8%). The most common grade ≥3 AE was thrombocytopenia and platelet count decrease (5.6%).
Overall, 87.3% of the treatment-related AEs were from lifirafenib and 88.7% were from mirdametinib. Serious AEs related to lifirafenib treatment were seen in 42.3% of patients and SAEs from mirdametinib were observed in 14.1%. DLT TEAEs occurred in 9.9% of patients overall. These TEAEs lead to dose modification in 57.7%, discontinuation of treatment in 5.6%, and death in 5.6%.
Based on these findings, Solomon concluded that the low-grade serous ovarian cancer, NSCLC, and endometrial cancer patient population who harbor BRAF and KRAS mutations may be sensitive to the combination of lifirafenib and mirdametinib. Further study on the benefit-risk profile of the combination is warranted. https://www.targetedonc.com/view/lifirafenib-plus-mirdametinib-shows-tolerable-safety-in-braf-kras-mutant-advanced-solid-tumors

[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 that is benzhydroxamic acid (N-hydroxybenzamide) in which the hydroxamic acid group has been converted to the corresponding 2,3-dihydroxypropyl ester and in which the benzene ring has been substituted at position 2 by a (2-fluoro-4-iodophenyl)amino group and at positions 3 and 4 by fluorines (the R enantiomer). It has a role as an EC 2.7.12.2 (mitogen-activated protein kinase kinase) inhibitor and an antineoplastic agent. It is a hydroxamic acid ester, a secondary amino compound, a member of monofluorobenzenes, an organoiodine compound, a member of propane-1,2-diols and a difluorobenzene.
PD-0325901 has been used in trials studying the treatment and basic science of Melanoma, Solid Tumour, Solid Tumors, Advanced Cancer, and Breast Neoplasms, among others.
Mirdametinib is an orally bioavailable, synthetic organic molecule targeting mitogen-activated protein kinase kinase (MAPK/ERK kinase or MEK) with potential antineoplastic activity. Upon administration, mirdametinib selectively binds to and inhibits MEK, which may result in the inhibition of the phosphorylation and activation of MAPK/ERK and the inhibition of tumor cell proliferation. The dual specific threonine/tyrosine kinase MEK is a key component of the RAS/RAF/MEK/ERK signaling pathway that is frequently activated in human tumors.

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A novel series of benzhydroxamate esters derived from their precursor anthranilic acids have been prepared and have been identified as potent MEK inhibitors. 2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide, 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 trial. CI-1040 suffered however from poor exposure due to its poor solubility and rapid clearance, and as a result, development of the compound was terminated. Optimization of the diphenylamine core and modification of the hydroxamate side chain for cell potency, solubility, and exposure with oral delivery resulted in the discovery of the clinical candidate N-(2,3-dihydroxy-propoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide PD 0325901.[1]

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Papillary thyroid carcinomas (PTC) are the most common type of thyroid malignancy. Most PTC carry one of the two mutations, RET/PTC rearrangement or BRAF mutation. Both mutations are able to activate the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling transduction pathway leading to cellular proliferation, differentiation, and apoptosis. PD0325901 is a specific MEK1/2 inhibitor and therefore is a promising drug to treat thyroid cancers with either RET/PTC or BRAF mutation. In this study we tested the effects of PD0325901 on PTC cells harboring either mutation in vitro by growth curves and Western blots and in vivo using a murine orthotopic xenograft model. We found that 50% growth inhibition (GI(50)) by PD0325901 was 11 nmol/L for the PTC cells with the RET/PTC1 rearrangement and 6.3 nmol/L for PTC cells with a BRAF mutation, with both concentrations readily achievable in serum. After 1 week of oral administration of PD0325901 (20-25 mg/kg/day) in mice, no tumor growth was detected in mice inoculated with PTC cells bearing a BRAF mutation. For PTC with the RET/PTC1 rearrangement, the average tumor volume of the orthotopic tumor was reduced by 58% as compared with controls. In conclusion, our data suggested that PTC cells carrying a BRAF mutation were more sensitive to PD0325901 than were PTC cells carrying the RET/PTC1 rearrangement. Our findings support the clinical evaluation of PD0325901 for patients with PTC and potentially other carcinomas with BRAF mutations.[3]

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.)