EPZ020411

Biological Activity

In vitro: Treatment with EPZ020411 results in a dose-dependent decrease in H3R2 methylation in A375 human melanoma cells exogenously overexpressing PRMT6(IC50=0.637±0.241 μM). In biochemical assays EPZ020411 is over 100-fold selective for PRMT6/8/1 compared to other histone methyltransferases including four arginine methyltransferases (PRMT3, PRMT4, PRMT5, and PRMT7). The compound shows poor permeability in the parallel artificial membrane permeation assay. In vivo: EPZ020411 shows good bioavailability following subcutaneous dosing in rats making it a suitable tool for in vivo studies. Male Sprague-Dawley rats administered a single dose of EPZ020411 at 1 mg/kg by i.v. bolus show a moderate clearance (CL) of 19.7±1.0 mL/min/kg, with a volume of distribution at steady state (Vss) of 11.1±1.6 L/kg, translating to a mean terminal half-life (t1/2) of 8.54±1.43 h. Following 5 mg/kg s.c. dosing, a good bioavailability of 65.6 ± 4.3% is observed, leading to EPZ020411 unbound blood concentration remaining above the PRMT6 biochemical IC50 value for more than 12 h.

Protocol (for reference only)

Cell Experiment
Cell lines	A375 cells
Preparation method	A375 (CRL-1619) cells are cultured in DMEM plus 10% (vol/vol) FBS. PRMT6 is cloned into BamHI and EcoRI sites of a pcDNA4 HisMAX_A plasmid. Transfection of his-tagged PRMT6 or vector control is carried out using Lipofectamine LTX and Plus reagent according to procedures recommended by the manufacturer. Cells are seeded at 200,000 cells/well in 6-well plates. The following day, the cells are concurrently transfected and treated with compound in 0.25% DMSO. Cells are incubated in the presence of increasing concentrations of compound up to 20 μM. Cell pellets are collected after 48 hours of compound treatment.
Concentrations	0-20 μM
Incubation time	48 h

Animal Experiment
Animal models	Sprague-Dawley rats
Formulation	saline, pH 6.5
Dosages	1 mg/kg
Administration	i.v.

Chemical Information

Molecular Weight	442.59
Formula	C25H38N4O3
CAS Number	1700663-41-7
Solubility (25°C)	DMSO 60 mg/mL
Storage	Powder          -20°C   3 years ;  4°C   2 years In solvent       -80°C   6 months ;  -20°C   1 month

Conversion of different model animals based on BSA (PMID: 27057123)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

References

[1] Mitchell LH, et al. ACS Med Chem Lett. Aryl Pyrazoles as Potent Inhibitors of Arginine Methyltransferases: Identification of the First PRMT6 Tool Compound.