Metarrestin (ML246) kills the perinuclear area of PC3M-GFP-PTB cells with an IC50 of 0.39 μM[2]. In several human skeletal lineages, metarrestin (1 μM; 24 h) lowers the nuclear compartment infection rate. Metarrestin Effects: PC3M and PANC1 cell proliferation is efficiently inhibited by metarrestin (0.6 μM; 24 hours); normal fibroblasts (GM02153) are not affected by this drug [2]. PC3M and PANC1 cell proliferation is significantly inhibited by metarrestin (1 μM; 24 hours) [2]. ] had no discernible effect on the levels of the Pol I large subunits RPA194 and UBF in the HeLa, PC3M, and PANC1 cell lines. In cells, metarrestin significantly lowers 5'ETS RNA [2].

Metarrestin (ML246) kills the perinuclear area of PC3M-GFP-PTB cells with an IC50 of 0.39 μM[2]. In several human skeletal lineages, metarrestin (1 μM; 24 h) lowers the nuclear compartment infection rate. Metarrestin Effects: PC3M and PANC1 cell proliferation is efficiently inhibited by metarrestin (0.6 μM; 24 hours); normal fibroblasts (GM02153) are not affected by this drug [2]. PC3M and PANC1 cell proliferation is significantly inhibited by metarrestin (1 μM; 24 hours) [2]. ] had no discernible effect on the levels of the Pol I large subunits RPA194 and UBF in the HeLa, PC3M, and PANC1 cell lines. In cells, metarrestin significantly lowers 5'ETS RNA [2].

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Metarrestin disassembles the perinucleolar compartment (PNC) in solid organ cancer cell lines at submicromolar concentrations, with a reported cell-based PNC disassembly IC50 of 0.4 µM. [1]
A BODIPY-labeled analog of Metarrestin (NCGC00387350) was able to disassemble PNC with an IC50 of 3.32 µM in PC3M cells and showed homogeneous intracellular accumulation in the cytosol when visualized by confocal microscopy. [1]
Cellular uptake studies in KPC and PC3M cells showed that cellular levels of Metarrestin reached 40–100% of media concentrations within 40 minutes after exposure to 1 µM or 10 µM of the drug, indicating rapid equilibrium. [1]
In murine (KPC) and human pancreatic cancer cell lines, treatment with increasing concentrations of Metarrestin for 24 hours led to a dose-dependent normalization of mRNA expression levels for FOXA1 and FOXO6, as determined by qRT-PCR. [1]
Incubation of CD-1 mouse and Sprague-Dawley rat hepatocytes with 10 µM Metarrestin for 4 hours yielded three oxidative metabolites at very low abundance (<5% compared to parent). The main metabolite was identified as a ketone derivative (keto-metarrestin), which was synthesized and shown to be equipotent to the parent compound in PNC disassembly assays. [1]

At 25 mg/kg, metorrestin (ML246; 5–25 mg/kg; intraperitoneally; once daily; for six weeks) demonstrated a decreased burden on the lungs and liver (p <0.01) [2]. meal; 70 ppm; 10 mg/kg) increases the NSG PANC1 pancreatic cancer metastatic model's longevity [2]. The growth of truck carcinoma metastases (PC3M) and the PDX mouse model of metastatic breast cancer is inhibited by metorrestin (5, 25 mg/kg; i.p.; extra 4 weeks) [2]. The half-life of metorrestin (5 and 25 mg/kg; IP) is 4.6 to 5.5 hours [2].

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In tumor-bearing KPC mice, a 14-day treatment with 25 mg/kg/day Metarrestin via oral gavage significantly reduced the prevalence of PNC structures in pancreatic tumors from 38.2% to 9.8%. [1]
The intratumor concentration of Metarrestin at 24 hours after a single oral dose of 25 mg/kg was high, with a mean value of 6.2 µg/g tissue (equivalent to 13 µM), well above the cell-based IC50 of 0.4 µM. [1]
Dose-dependent normalization of FOXA1 and FOXO6 mRNA expression, as measured by qRT-PCR, was observed in KPC tumors from mice treated with Metarrestin, and the expression levels of these genes correlated with the intratumor drug exposure (AUC0-24h). [1]
Previous anti-metastasis and survival studies in mice (referenced within the text but not detailed in this PK study) identified an efficacious dose range of 10-25 mg/kg. [1]

PNC Disassembly Assay (High-Content Imaging): The assay for PNC disassembly has been previously described. Briefly, cancer cells (e.g., PC3M expressing GFP-tagged PTB1) are cultured in multi-well plates. Cells are treated with test compounds like Metarrestin or its analogs for a defined period (e.g., 24 hours). After fixation, cells are stained with Hoechst 33342 to visualize nuclei. The disassembly of fluorescently labeled PNC structures is quantified using high-throughput immunofluorescence microscopy imaging. The concentration causing 50% PNC disassembly (IC50) is determined. [1]
Cellular Drug Uptake and Concentration Measurement: KPC or PC3M cells are exposed to defined concentrations of Metarrestin (e.g., 1 µM or 10 µM) in culture media. At specified time points, cells are harvested. Cellular concentrations of Metarrestin are then quantified using UPLC-MS/MS. [1]
Gene Expression Analysis (qRT-PCR): Total RNA is isolated from treated cells or tissues. cDNA is synthesized using reverse transcriptase. Target gene mRNA levels (e.g., FOXA1, FOXO6) are quantified by real-time PCR using specific primers, with normalization to reference genes (e.g., GAPDH). [1]

Animal/Disease Models: NOD/IL2 gamma (null) PANC1 mouse primary tumor tissue [2]
Doses: 5 and 25 mg/kg
Route of Administration: IP; one time/day; for six weeks
Experimental Results: At the 25 mg/kg dose, Both liver (p <0.01) and lung metastatic burden were diminished. demonstrated a Dramatically diminished prevalence of perinucleolar compartments in metastatic and primary tumor tissue.

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Animal/Disease Models: Female balb/c (Bagg ALBino) mouse [2] Doses: 5 and 25 mg/kg (pharmacokinetic/PK/PK analysis)
Route of Administration: IP
Experimental Results: Good in vivo exposure, distribution and tolerance, with a half-life of 4.6 to 5.5 hrs (hrs (hours)) .

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Single-Dose PK Study in C57BL/6 Mice: Metarrestin was formulated in a solution of 30% PEG-400 and 70% (20% w/v HP-β-CD in water). It was administered to 6-8-week-old male C57BL/6 mice via a single intravenous (IV) tail vein injection at 3 mg/kg (3 mL/kg dosing volume) or via oral gavage (PO) at 3 and 10 mg/kg (10 mL/kg dosing volume). Blood and liver samples were collected at multiple time points post-dosing (n=3 per time point). [1]
Single and Multiple-Dose PK Study in KPC Mice: Tumor-bearing KPC mice (with pancreatic tumor volume 100-250 mm³) received Metarrestin formulated as above. For single-dose (SD) PK, a 25 mg/kg dose was administered via PO gavage. For multiple-dose (MD) PK, a daily dose of 25 mg/kg was administered via PO gavage for 14 days. Blood, pancreatic tumor, spleen, and liver samples were collected at scheduled times on Day 1 (SD) or Day 14 (MD). Dosing solutions were prepared fresh on the day of administration. [1]
Steady-State PK Study via Medicated Chow: KPC mice were fed with Metarrestin-infused rodent diet (formulated at 70 ppm, targeting a ~10 mg/kg/day dose based on food consumption) for 10 days. On Day 10, while animals remained on the medicated chow, blood and tissue samples were collected every 3 hours over a 24-hour period to assess steady-state concentrations. [1]

Absorption: Metarrestin was well absorbed after oral administration in mice. The oral bioavailability of C57BL/6 mice was greater than 80% after a single dose. The time to reach maximum plasma concentration (Tmax) was 2 hours for low doses (3, 10 mg/kg, orally) and 6 hours for high doses (25 mg/kg, orally). [1] Distribution: After intravenous injection of 3 mg/kg in C57BL/6 mice, Metarrestin showed a large volume of distribution (Vdss = 17 L/kg) at steady state, indicating its extensive tissue distribution. Tissue concentrations were significantly higher than plasma concentrations. After a single oral dose of 25 mg/kg in KPC mice, the tissue/plasma ratios at AUC0-24h were: tumor 19, spleen 33, liver 44. After multiple administrations over 14 days (25 mg/kg/day, orally), these ratios were: tumor 37, spleen 30, liver 31. After repeated administration, the drug accumulated in tissues, with MD:SD AUC0-24h ratios of 5.0 for tumors, 2.4 for spleens, and 1.8 for livers. [1] Metabolism: Incubation with mouse and rat hepatocytes showed that Metarrestin underwent oxidative metabolism, producing at least three minor metabolites, the major of which were ketone derivatives. These metabolites were present in very low abundance (<5% of the parent compound). The ketone metabolite exhibited PNC depolymerization activity similar to that of the parent compound. [1] Excretion (clearance): After intravenous injection of 3 mg/kg into C57BL/6 mice, the plasma clearance (CLp) was 48 mL/min/kg, which is moderate. [1]
Major pharmacokinetic parameters in mice: In C57BL/6 mice (3 mg/kg intravenously): plasma clearance (CLp) = 48 mL/min/kg, steady-state volume of distribution (Vdss) = 17 L/kg, and terminal half-life (t1/2) approximately 5 hours. In C57BL/6 mice (3 and 10 mg/kg orally): bioavailability >80%, peak plasma concentration (Cmax) 55.7 ng/mL and 349 ng/mL, respectively, and area under the curve (AUC0-∞) 871 and 4790 ng·h/mL, respectively. In KPC mice (25 mg/kg orally once daily): peak plasma concentration (Cmax) = 810 ng/mL, AUC0-∞ = 14,400 ng·h/mL, and terminal half-life (t1/2) = 8.5 hours. Under steady-state conditions, after feeding with medicated feed (approximately 10 mg/kg/day), the average plasma concentration was approximately 0.2 µM, and the average tumor concentration was approximately 4.0 µM. [1]

In KPC mice, oral administration of 25 mg/kg of Metarrestin for 14 consecutive days did not result in changes in body weight, decreased activity, or abnormal liver function compared to the vector control group. No toxicity was observed in mice within the effective dose range of 5 to 25 mg/kg (as previously described, referencing previous studies). [1]

[1]. Pharmacokinetic evaluation of the perinucleolar compartment disassembler metarrestin in wild-type and Pdx1-Cre;LSL-KrasG12D/+;Tp53R172H/+ (KPC) mice, a genetically engineered model of pancreatic cancer. Cancer Chemother Pharmacol. 2018 Dec;82(6):1067-1080.

[2]. Metarrestin, a perinucleolar compartment inhibitor, effectively suppresses metastasis. Sci Transl Med. 2018 May 16;10(441).

Metarrestin is an oral small molecule perinuclear compartment (PNC) inhibitor with potential antitumor activity. Although the exact mechanism of action of the drug has not been fully elucidated, after oral administration, Metarrestin disrupts the structure of the PNC and inhibits the transcription of RNA polymerase (Pol) I. This leads to a reduction in the amount of PNC in cancer cells, thereby inhibiting tumor growth and spread. The PNC is a subnuclear structure and a phenotypic marker of metastatic cancer cells. High levels of PNC are associated with disease progression and poor patient prognosis. Metarrestin (ML-246) is a first-in-class small molecule clinical candidate that was discovered through high-throughput screening aimed at finding compounds that can disrupt the perinuclear compartment (PNC), a subnuclear structure specific to metastatic cancer cells. [1] Its mechanism of action involves the disruption of the PNC, and the disruption of the PNC is associated with the metastatic phenotype. The drug's mechanism of action is cytosuppression rather than cytotoxicity, because the PNC reassembles and cell proliferation is restored after the drug is removed in vitro. [1]
This compound is being developed as an anti-metastatic therapeutic. Its favorable pharmacokinetic characteristics, including high oral bioavailability, excellent tissue penetration (even into poorly vascularized pancreatic tumors), and the lack of toxicity observed at effective doses in mice, support its further development. [1]

C31H30N4O

474.60

474.24

C, 78.45; H, 6.37; N, 11.81; O, 3.37

1443414-10-5

50985821

White to off-white solid powder

5.6

2

3

5

36

759

0

WSMXAUJFLWRPNT-UHFFFAOYSA-N

InChI=1S/C31H30N4O/c32-30-28-27(23-12-6-2-7-13-23)29(24-14-8-3-9-15-24)34(20-22-10-4-1-5-11-22)31(28)33-21-35(30)25-16-18-26(36)19-17-25/h1-15,21,25-26,32,36H,16-20H2

4-(7-benzyl-4-imino-5,6-diphenylpyrrolo[2,3-d]pyrimidin-3-yl)cyclohexan-1-ol

ML246 ML-246

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)

DMSO : ~31.25 mg/mL (~65.84 mM)

Solubility in Formulation 1: 2.08 mg/mL (4.38 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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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 (Please use freshly prepared in vivo formulations for optimal results.)