Ibrutinib

Ibrutinib shows the potent and irreversible inhibitory effect and selectivity for Btk enzymatic activity. In BCR pathway-activated DOHH2 cell line, Ibrutinib inhibits autophosphorylation of Btk, phosphorylation of Btk's physiological substrate PLCγ, and phosphorylation of further downstream kinase, ERK with IC50 of 11 nM, 29 nM and 13 nM, respectively. ^[1] Ibrutinib exhibits a significant dose-dependent and time-dependent induction of cytotoxicity in chronic lymphocytic leukemia (CLL) cells. In addition, Ibrutinib induces cell death depending on caspase pathway activation and antagonizes the ability of CLL cells to proliferate after TLR signaling. ^[2] A recent study shows that Ibrutinib inhibits BCR-activated primary B cell proliferation with IC50 of 8 nM and results in inhibition of TNFα, IL-1β and IL-6 production in primary monocytes with IC50 of 2.6 nM, 0.5 nM and 3.9 nM, respectively. ^[3]

In a collagen-induced arthritis model, Ibrutinib significantly reduces clinical arthritis scores reflecting paw swelling and joint inflammation by inhibiting B-cell activation. In a MRL-Fas(lpr) lupus model, Ibrutinib reduces renal disease and autoantibody production. ^[1] In an adoptive transfer TCL1 mouse model of CLL, Ibrutinib (25 mg/kg/day) causes a transient early lymphocytosis, and delays CLL disease progression. ^[4]

• Kinase Assays

  In vitro kinase IC50 values are measured using ³³P filtration binding assay after 1 hour incubation of kinase, ³³P-ATP, Ibrutinib, and substrate [0.2 mg/mL poly(EY)(4:1]. Assays are performed at Reaction Biology.

• Cell lines

  Chronic lymphocytic leukemia (CLL) cells

• Concentrations

  0.01-100 μM

• Incubation Time

  48 hours

• Method

  MTT (3'[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assays are performed to determine cytotoxicity. Briefly, cells (CLL B cells or healthy volunteer T cells or NK cells) are incubated for 48 hours with different concentrations of Ibrutinib, or vehicle control. MTT reagent is then added, and plates are incubated for an additional 20 hours before washing with protamine sulfate in phosphate-buffered saline. Dimethyl sulfoxide is added, and absorbance is measured by spectrophotometry at 540 nm in a Labsystems plate reader. Cell viability is also measured at various time points with the use of annexin/PI flow cytometry. Data are analyzed with Expo-ADC32 software package. Results are expressed as the percentage of total positive cells over untreated control. Experiments examining caspase-dependent apoptosis includes the addition of 100μM Z-VAD.

• Animal Models

  MRL-Fas(lpr) lupus model and collagen-induced arthritis model.

• Dosages

  ≤50 mg/kg

• Administration

  Administered via p.o.