INCB18424

Biological Activity

INCB18424 (Ruxolitinib), the most advanced JAK-1 and JAK-2 inhibitor, is in Phase III testing to treat myelofibrosis. INCB18424 demonstrated potent in vitro cellular activity (100-130 nM) in an engineered Ba/F3 cell line harboring the JAK2V617F mutation and EPO-independent erythroid colony growth of precursor cells from PV patients (IC50 of 67 nM) while showing lesser effect on normal colony formation from healthy donors (IC50 > 400 nM).

Product Citations

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  Front Immunol. 2022 May 23;13:866638.

  A Novel STAT3 Gain-of-Function Mutation in Fatal Infancy-Onset Interstitial Lung Disease
  INCB18424 purchased from AbMole

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  BMC Biol. 2021 May 20;19(1):108.

  Very long intergenic non-coding (vlinc) RNAs directly regulate multiple genes in cis and trans
  INCB18424 purchased from AbMole

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  Blood Adv. 2020 Jul 14;4(13):3000-3010.

  Inflammation-driven activation of JAK/STAT signaling reversibly accelerates acute myeloid leukemia in vitro
  INCB18424 purchased from AbMole

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  J Virol. 2020 Feb 14;94(5):e01791-19.

  Targeting Kaposi's Sarcoma-Associated Herpesvirus ORF21 Tyrosine Kinase and Viral Lytic Reactivation by Tyrosine Kinase Inhibitors Approved for Clinical Use
  INCB18424 purchased from AbMole

Customer Product Validations & Biological Datas

	Source	Leukemia (2013). Figure 2. INCB18424
	Method	MTS assay
	Cell Lines	leukemia cells
	Concentrations	1 μM
	Incubation Time	48 h
	Results	INCB18424 showed a significantly better inhibitory effect than TG101209 at concentrations below 1 μM. Both inhibitors also promoted apoptosis (Figure 2b) and delayed cell cycle progression (Figure 2c) of the AEtr leukemia cell line.

	Source	Leukemia (2013). Figure 1. INCB18424
	Method	CFU assays
	Cell Lines	AML1–ETO- and AE9a-transformed cells
	Concentrations	150, 300, 600, 1200 nM
	Incubation Time	36 h
	Results	Similar to the shRNA results, both inhibitors reduced the colony-forming ability of the transformed cells in a dose-dependent manner

Protocol

Cell Experiment
Cell lines	Ba/F3-EpoR-JAK2V617F and HEL cell lines
Preparation method	Cell proliferation assay. Cells were seeded at 2000/well of white bottom 96-well plates, treated with compounds from DMSO stocks (0.2% final DMSO concentration), and incubated for 48 hours at 37°C with 5% CO2. Viability was measured by cellular ATP determination using the Cell-Titer Glo (Promega) luciferase reagent or viable cell counting. Values were transformed to percent inhibition relative to vehicle control, and IC50 curves were fitted according to nonlinear regression analysis of the data using PRISM GraphPad.
Concentrations	0~10 µ M
Incubation time	48 h

Animal Experiment
Animal models	myeloproliferative neoplasm mouse model
Formulation	5% dimethyl acetamide, 0.5% methocellulose
Dosages	180mg/kg began within 24 hours of cell inoculation, twice daily
Administration	oral gavage

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

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.

Chemical Information

References

[1] Chase A, et al. Haematologica. Ruxolitinib as potential targeted therapy for patients with JAK2 rearrangements.

[2] Mascarenhas J, et al. Curr Med Chem. Biology and Clinical Management of Myeloproliferative Neoplasms and Development of the JAK Inhibitor Ruxolitinib.

[3] Eghtedar A, et al. Blood. Phase 2 study of the JAK kinase inhibitor ruxolitinib in patients with refractory leukemias, including postmyeloproliferative neoplasm acute myeloid leukemia.

[4] Harrison C, et al. N Engl J Med. JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis.