Trifluridine/tipiracil hydrochloride mixture (TAS-102) is a novel oral combination drug containing trifluridine (TFT) and Tipiracil hydrochloride (TTP) in a molar ratio of 2:1.

Trifluridine/tipiracil hydrochloride mixture是一种口服复合化合物，由基于胸苷的核苷类似物三氟脱氧尿苷(FTD)和提拉吉尔 hydrochloride (TPI)组成[1]。三氟脱氧尿苷的磷酸化形式被并入DNA，导致DNA功能障碍和细胞周期阻滞。胸苷磷酸化酶抑制剂阻止FTD的降解并抑制血管生成。因此，Trifluridine/tipiracil hydrochloride mixture治疗导致大量三氟脱氧尿苷并入DNA，并激活涉及Chk1磷酸化和G2/M期间细胞周期阻滞的类似DNA损伤响应途径[2]。

FTD经静脉注射给予人体后，其消除半衰期非常快速（18分钟），这是因为FTD迅速降解为其主要代谢产物5-三氟甲基-2,4(1H,3H)-嘧啶二酮所致。在猴子中，仅通过口服给药，FTD在血浆中的浓度非常低，这表明FTD在肝脏和肠道经由TP酶的一次性代谢作用非常彻底。然而，加入TPI(盐酸替匹拉酯)可以实现口服给药。通过抑制TP，TPI在口服给药后抑制了FTD在肝脏和肠道中的降解，从而提高了其生物利用度。TP酶促进了如FTD这样的嘧啶2'-脱氧核苷的磷酸解反应。使用人类CRC肿瘤移植模型的小鼠进行的研究确定，最大抗肿瘤活性是以1:0.5的摩尔比达到的，并且在小鼠和猴子中的研究显示，几乎用相同的比例达到了FTD的最大血浆浓度。此外，这一比例在抗肿瘤活性和毒性之间产生了一个有利的平衡。与单独给予FTD相比，小鼠在TPI共同给药时观察到的毒性较低。Trifluridine/tipiracil hydrochloride mixture（FTD）可以克服对5-FU的获得性抗性，因为Trifluridine/tipiracil hydrochloride mixture的主要作用机制与5-FU的主要代谢酶如TS和OPRT无关。Trifluridine/tipiracil hydrochloride mixture在5-FU耐药的癌症中显示出了疗效[1]。

IC50 determination of compounds against EGFR enzymes: The inhibition potency of compounds against EGFR WT and mutant enzymes is assessed using CisBio homogenous time resolved fluorescence approach (HTRF, Cat No. 62TK0PEJ) according to manufacturer's instruction. The final enzyme concentrations used in this assay are 0.1 nM, 0.03 nM, and 0.026 nM for EGFR wild type, L858R and Exon19Del, respectively, and 0.8 μM, 4 μM and 25 μM ATP, corresponding to the Km values of EGFR enzymes, are applied accordingly. In brief, 3 μL of ATP and 2 μM TK biotin-peptide substrate are incubated in the presence or absence of serially diluted compound at room temperature in 384-well Greiner white polystyrene assay plates. The reaction is initiated by addition of 3 μL kinase which could phosphorylate the substrate peptide, and the assay buffer contains 1 mM DTT, 5 mM MgCl2, 1 mM MnCl2, and 0.01% CHAPS. After 30 minutes incubation, the reaction is stopped by the addition of 6 μl detection reagent mix containing 250 nM Strep-XL665 and TK Ab Europium Cryptate diluted in detection buffer. The plates are incubated for 1 h before the fluorescence is then measured at 615 nm and 665 nm, respectively with excitation wavelength at 320 nm by EnVision Multilabel Reader from Perkin Elmer using standard HTRF settings. The calculated signal ratio of 665 nm/615 nm is proportional to the kinase activity. The concentration of compound producing 50% inhibition of the respective kinase (IC50) is calculated using four-parameter logistic fit.