Uses of ibrutinib
A technology of ibrutinib and uses, applied in a new use field of ibrutinib, can solve the problems of poor EGFR mutant selectivity, patient recurrence, and low clinically tolerated dose of the drug
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Embodiment 1
[0041] Example 1: Effect of Ibrutinib (PCI-32765) on Cancer Cell Growth
[0042]By testing the effect of ibrutinib (PCI-32765) on cancer cell growth, we further evaluated the selectivity of ibrutinib (PCI-32765) in inhibiting cancer cell proliferation. In this example, we selected human non-small cell lung cancer cell NCI-H1975 (expressing EGFR L858R / T790M double mutant gene), human lung adenocarcinoma cell NCI-H3255 (expressing EGFR L858R mutant gene), mouse EGFR-T790M BaF3 cells (stable expression of TEL-EGFR-T790M activated mutant kinase), human non-small cell lung cancer cell PC-9 (expression of EGFR delE746_A750 mutant gene), human skin squamous cell carcinoma A431 (expression of wild-type EGFR gene), human non-small cell lung cancer cells Small cell lung cancer cell NCI-H460 (expressing wild-type EGFR gene), human lung adenocarcinoma cell A549 (expressing wild-type EGFR gene), human non-small cell lung cancer cell NCI-H358 (expressing wild-type EGFR gene). The above c...
Embodiment 2
[0046] Example 2: In vitro inhibitory activity (enzyme activity) of ibrutinib (PCI-32765)
[0047] The IC50 values of ibrutinib (PCI-32765) on EGFR (WT), EGFR / T790M, and EGFR L858R / T790M were determined in the in vitro enzyme activity assay as described below. The intracellular segment (699-1068) of EGFR was cloned into the insect expression vector pAcG2T, using the insect expression system BaculoGold TM Protein expression was performed using the Baculovirus Expression System (BDPharmingen) with a GST tag. At the same time, the T790M and L858R sites were mutated to obtain the T790M single mutation vector and the T790M / L858R double mutation vector of EGFR respectively. The constructed vector was transfected into the SF9 packaging virus, and the virus was used to infect the SF9 expression protein.
[0048] Take 9 μL (6 ng / μL) of purified EGFR (WT), EGFR (T790M), EGFR (T790M / L858R) protein kinase and react with three-fold serially diluted drugs WZ4002 and PCI-327651 μL at ...
Embodiment 3
[0053] Embodiment 3: plate colony formation experiment
[0054] The NCI-H1975 human non-small cell lung cancer cells in the exponential growth phase carrying the EGFR L858R / T790M double mutation were made into a cell suspension by conventional trypsinization and passaging. Pipette the cell suspension repeatedly to fully disperse the cells. NCI-H1975 human non-small cell lung cancer cells carrying EGFR L858R / T790M double mutation were counted, and the cell concentration was adjusted with medium.
[0055] According to the proliferation ability of NCI-H1975 human non-small cell lung cancer cells carrying EGFR L858R / T790M double mutation, according to 10 5 The concentration of cells / well was inoculated into a six-well plate (4 cm in diameter) containing 2 mL of medium, and the culture plate was gently shaken in a cross direction to make the cells evenly dispersed.
[0056] Place the Petri dish at 37°C, 5% CO 2 After 24 hours of incubation in medium, the drug concentration was...
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