Idelalisib (GS-1101) is a small molecule inhibitor of the PI3K catalytic subunit p110δ (IC50: 2.5 nM). The selectivity for p110δ is 40- to 300-fold than p110α/β/γ.

p110γ:89 nM (cell free), p110β:565 nM (cell free), p110δ:2.5 nM (cell free)

Idelalisib 是一种口服的 p110δ 抑制剂，目前正在对 B 细胞恶性肿瘤患者进行临床评估。Idelalisib 对 p110δ 的选择性比对其他 PI3K 类 I 酶（IC50 p110δ = 2.5nM; p110α、p110β 和 p110γ 的 IC50 分别为 820、565 和 89nM）高出 40 至 300 倍。相对于 C2β、hVPS34、DNA-PK 和 mTOR 等相关激酶，其选择性更高（400 至 4000 倍），而在 10μM 浓度下对一个包含 402 种多样激酶的测试组未显示任何活性[1]。Idelalisib 能够在剂量和时间依赖的方式下促进原发性 CLL 细胞的凋亡，这一过程与常见的预后标记物无关。Idelalisib 引发的细胞毒性依赖于半胱天冬酶，并且通过在基质细胞上共培养并未减弱[2]。CAL-101 抑制 CLL 细胞向 CXCL12 和 CXCL13 的趋化以及在基质细胞下的迁移（伪包裹现象）。Idelalisib 还能下调在基质共培养和 BCR 触发后的趋化因子的分泌。Idelalisib 减少了来自 BCR 或像护士一样的细胞的生存信号，并抑制 BCR- 和趋化因子受体诱导的 AKT 和 MAP 激酶（ERK）活化[3]。

单次静脉注射40 mg/kg的Idelalisib，于缺血前15分钟给药（预处理），在野生型小鼠中显著降低了72小时后的梗塞面积。然而，较低剂量elalisib（20、10和1 mg/kg）未能实现显著的保护效果。重要的是，即便在再灌注开始后3小时给药（治疗后），每千克体重40 mg的Idelalisib剂量仍能有效减少梗塞体积，与对照组相比平均降低了44%[4]。

PI3K assay was performed on whole-cell lysates from CLL or normal B cells. A PI3K ELISA assay was performed according to the manufacturer's instructions. Briefly, whole-cell extracts were added to a mixture of PI(4,5)P2 substrate and reaction buffer containing adenosine triphosphate (ATP) and allowed to incubate at room temperature. The reaction was stopped by adding PI(3,4,5)P3 detector mixed with EDTA (ethylenediaminetetraacetic acid) and allowed to incubate at room temperature for 1 hour. After this time, the mixture was transferred from each well to a PI3K ELISA plate and allowed to incubate 1 hour. Plates were washed and then incubated with a secondary detector for 30 minutes. Plates were washed again, and 3,3′,5,5′-tetramethylbenzidine solution was added for 5 minutes at which time H2SO4 was added to stop all reactions. Plates were read at 450 nm on a Labsystems 96-well plate reader [2].

MTT assays were performed to determine cytotoxicity. Briefly, 1 × 10^5 cells (CLL B cells or healthy volunteer T cells or NK cells) were incubated for 48 hours with different concentrations of CAL-101, 25μM LY294002, or vehicle control. MTT reagent was then added, and plates were incubated for an additional 20 hours before washing with protamine sulfate in phosphate-buffered saline. Dimethyl sulfoxide was added, and absorbance was measured by spectrophotometry at 540 nm in a Labsystems plate reader. Cell viability was also measured at various time points with the use of annexin/PI flow cytometry. Data were analyzed with Expo-ADC32 software package. At least 10 000 cells were counted for each sample. Results were expressed as the percentage of total positive cells over untreated control. Experiments examining caspase-dependent apoptosis included the addition of 100μM Z-VAD. Experiments examining survival signals included the addition of 1 μg/mL CD40L, 800 U/mL IL-4, 50 ng/mL BAFF, 20 ng/mL TNF-α, or coculturing on fibronectin or stromal (HS-5 cell line) coated plates. Stromal coculture was done by plating a 75-cm2 flask (80%-100% confluent) per 6-well plate 24 hours before the addition of CLL cells [2].

For Idelalisib (CAL-101) treatment, wild-type C57BL/6 mice were administered either 40 mg kg 1 CAL-101 or vehicle DMSO, by 25 ml infusion into the femoral vein, 15 min before I/R (pre-treatment), or 3 and 6 h after initiation of reperfusion (post-treatment). Controls and animals treated with CAL-101 underwent cerebral blood flow (CBF) measurements using a laser Doppler perfusion monitor. The CBF measurements obtained immediately before and after MCAO and again at 3 h after reperfusion showed a B90–95% reduction in the blood flow to the MCAO infarct region, which did not differ between groups [4].