Ibrutinib (PCI-32765) is a Bruton's tyrosine kinase (BTK) inhibitor (IC50=0.5 nM) with irreversible and selective properties. Ibrutinib blocks BTK to inhibit the proliferation and survival of B cells, and possesses antitumor activity, which can be used for the treatment of chronic lymphocytic leukemia, among others.

BMX:0.8 nM (cell free), CSK:2.3 nM (cell free), BTK:0.5 nM (cell free), BLK:0.5 nM (cell free), Brk:3.3 nM (cell free), FGR:2.3 nM (cell free)

方法：人 B 细胞淋巴瘤细胞 DOHH2 用 Ibrutinib (0.00064-2 µM) 孵育 1 h，再用 anti-IgG F(ab′)2 (30 µg/mL) 刺激 2 min，使用 Western Blot 检测靶点蛋白表达水平。
结果：Ibrutinib 抑制 Btk 的自体磷酸化 (IC50=11 nM)，Btk 的生理底物 PLCγ 的磷酸化 (IC50=29 nM)，和进一步的下游激酶 ERK 的磷酸化 (IC50=13 nM)。[1]
方法：原代人类B淋巴细胞用 Ibrutinib (1-1000 nM) 处理 30 min，然后用 anti-IgM F(ab')2 (10 µg/mL)、anti-CD3/CD28 (5 µg/mL) 或 PMA (0.5 µg/mL) 刺激细胞 72 h，使用 Cell Titer Glo reagent 检测细胞增殖。
结果：Ibrutinib 剂量依赖性地抑制抗 IgM 刺激的 B 淋巴细胞增殖 (IC50=8 nM)，但不抑制 PMA 刺激的增殖，PMA 激活PKC途径。[2]

方法：为检测体内抗炎活性，将 Ibrutinib (3.125-50  mg/kg) 灌胃给药给关节炎 DBA/1 小鼠，每天一次，持续十一天。
结果：在所有剂量下治疗的小鼠中观察到临床关节炎评分的显著抑制。在分别以 3.125 和 12.5 mg/kg/天 的剂量治疗 9 至 11 天后，疾病的临床症状出现部分和几乎完全消除。与体内抑制 B 细胞活化一致，抗胶原自身抗体的产生显著减少，总 IgG 水平适度降低。[1]

In vitro kinase IC50s were measured using 33P filtration binding assay after 1 h incubation of kinase, 33P-ATP, inhibitor, and substrate [0.2 mg/mL poly(EY)(4:1]. Assays were performed at Reaction Biology [1].

CD20+ B and CD3+ T cells were purified by negative selection (RosetteSep, >90% purity) from buffy coat PBMCs and viably frozen in 10% DMSO. Cells were thawed at 37 °C and maintained in growth media (RPMI media containing 10% FCS). B cells were stimulated with goat anti-human IgM F(ab′)2 (10 μg/mL) and T cells were stimulated with anti-CD3/CD28 coated beads at a 1:1 bead/cell ratio. Cells were stained with PE-CD69 and analyzed by flow cytometry, gating on viable lymphocytes. PCI-32765 at concentrations lower than 10 μM did not decrease B- or T-cell viability during the course of the experiment, although PCI-32765 did block the modest survival benefit of anti-IgM stimulation in B cells. For washout experiments, cells were rinsed three times in 10 volumes of growth media, a protocol that was confirmed to completely wash away inhibition of BCR signaling by PCI-29732, a reversible Btk inhibitor [1].

ale DBA/1 mice were immunized with type II collagen plus Freund adjuvant and boosted 21 d later. On a rolling basis, as significant swelling appeared in at least one paw, mice were enrolled and randomized. PCI-32765 or dexamethasone (0.2 mg/kg) was administered orally once per day for 11 d. Arthritis scores (0–5) were assigned to the mice based on the degree and extent of paw swelling. Mouse anti-type II collagen antibody and total IgG levels were measured by ELISA. Female MRL/MpJ-Faslpr mice received PCI-32765 by oral gavage once per day from week 8 through week 20. Proteinuria was monitored weekly. At week 20, serum was collected and analyzed for BUN and mouse anti-dsDNA antibody levels. Kidney histology was scored according to established criteria (26). No drug-induced weight loss was observed at any of the dose levels tested. These studies were carried out at Boulder Biopath according to approved animal care protocols. Results are presented as the mean ± SEM. Statistical significance between groups were evaluated with repeated measures one-way ANOVA or one-way ANOVA using GraphPad Prism with Tukey or Bonferroni multicomparison posttest [1].