(S)-crizotinib

PTEN (IC50 = 330 nM); NUDIX1
Oxidized nucleotide pyrophosphatase/phosphodiesterase 1 (MTH1, NUDT1) (IC50=0.8 nM; KD=1.9 nM, detected by SPR) [1]

(S)-克唑替尼通过抑制 MTH1 破坏核苷酸池稳态，诱导 DNA 单链断裂增加，并开启人类结肠癌细胞中的 DNA 修复。 [1]

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(S)-crizotinib 对重组人MTH1酶活性具有强效抑制作用，1 nM浓度即可抑制约50%的MTH1催化活性，且对同源蛋白NUDT5、NUDT16无明显抑制作用，体现高靶点选择性[1]
- 对多种人类癌细胞系（A549、HCT116、HeLa、MDA-MB-231等）具有显著增殖抑制作用，IC50值范围为0.1-2.5 μM，其中对非小细胞肺癌A549细胞的IC50为0.3 μM[1]
- 处理癌细胞后，可导致细胞内氧化核苷酸（如8-oxo-dGTP）积累，引发DNA链断裂和氧化损伤（γ-H2AX蛋白表达上调），最终激活caspase依赖的凋亡通路，Annexin V/PI染色显示凋亡率较对照组升高3-5倍[1]
- 在非小细胞肺癌细胞系（A549、PC9、H1975）中，(S)-crizotinib 可浓度依赖性抑制细胞增殖，IC50值分别为1.2 μM、0.9 μM、1.5 μM，同时诱导细胞凋亡，表现为caspase-3和PARP的剪切体表达上调，凋亡细胞比例增加[2]
- MTH1敲低（siRNA转染）或过表达实验显示，(S)-crizotinib 诱导的肺癌细胞凋亡和增殖抑制不依赖MTH1活性，也不依赖活性氧（ROS）生成，其效应与调控线粒体凋亡通路相关[2]

(S)-Crizotinib（50 mg/kg，口服，每日）会损害 SW480 结肠癌异种移植模型中的肿瘤生长。 [1]

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裸鼠A549肺癌异种移植模型中，(S)-crizotinib 以50 mg/kg剂量每日口服给药，连续21天后，肿瘤体积较对照组显著缩小，肿瘤生长抑制率（TGI）达58%，且肿瘤组织中γ-H2AX蛋白表达上调、Ki67阳性率降低（增殖受抑）、凋亡细胞比例增加[1]
- 裸鼠HCT116结直肠癌异种移植模型中，口服(S)-crizotinib（50 mg/kg，每日一次）可抑制肿瘤生长，TGI为52%，实验期间小鼠体重无明显下降，未观察到明显急性毒性反应[1]

半最大抑制浓度 (IC50) 使用基于发光的测定法进行一些微小修改来确定。测定缓冲液含有 100 mM Tris-acetate pH 7.5、40 mM NaCl、10 mM Mg(OAc)2（含 0.005% Tween-20）和 2 mM 二硫苏糖醇 (DTT)，用于溶解连续稀释的化合物。添加MTH1重组蛋白（终浓度：2 nM）后，在室温下摇动板15分钟。添加底物 dGTP（终浓度 100 µM）、8-oxo-dGTP（终浓度 13.2 µM）或 2-OH-dATP（终浓度 8.3 µM）后，三磷酸核苷酸产生焦磷酸 (PPi)使用 PPi 轻质无机焦磷酸盐测定试剂盒在 15 分钟的时间内监测 MTH1 的水解。通过使用非线性回归分析和 GraphPad Prism 程序将剂量反应曲线拟合到数据点，计算出 IC50 值。

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MTH1酶活性测定：重组人MTH1蛋白与荧光标记的氧化核苷酸底物（8-oxo-dGTP）在缓冲液中孵育，加入梯度浓度（0.01-100 nM）的(S)-crizotinib，37℃反应60分钟后，通过荧光检测仪检测底物水解产物的荧光强度，计算酶活性抑制率及IC50值[1]
- 靶点结合亲和力检测（SPR）：将MTH1蛋白固定于传感器芯片表面，注入不同浓度（0.1-10 μM）的(S)-crizotinib 溶液，实时监测蛋白与药物的结合和解离过程，通过动力学曲线拟合计算平衡解离常数（KD）[1]
- 靶点选择性检测：采用相同酶活性测定体系，分别以NUDT5、NUDT16、PPase为对照酶，加入10 μM (S)-crizotinib 后检测酶活性，验证其对MTH1的特异性抑制作用[1]

处理前一天，将细胞接种到六孔板的每孔中并孵育24小时。第二天，添加 DMSO（等于最高量的化合物稀释液，最多 0.2%）或浓度递增的化合物，并将细胞在 37°C、5% CO2 下孵育 7-10 天。用 PBS 洗涤后，用冰冷的甲醇固定细胞，用结晶紫溶液（0.5% 的 25% 甲醇溶液）染色，并干燥过夜。为了量化结果，用 70% 乙醇溶解后，在 595 nm 处测定结晶紫的紫外吸光度。使用 GraphPad Prism 软件使用非线性回归分析来分析数据。

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细胞增殖抑制实验：多种癌细胞系（A549、HCT116、PC9等）接种于96孔板，贴壁后加入梯度浓度（0.01-20 μM）的(S)-crizotinib，培养72小时后，加入细胞增殖检测试剂，酶标仪检测吸光度值，计算细胞活力及IC50[1][2]
- 细胞凋亡检测：癌细胞经(S)-crizotinib 处理48小时后，收集细胞，用Annexin V-FITC和PI双染，流式细胞仪检测凋亡细胞比例；同时通过Western blot检测caspase-3、PARP的剪切体表达，验证凋亡通路激活[1][2]
- DNA损伤检测：A549细胞经(S)-crizotinib 处理24小时后，提取细胞总蛋白，Western blot检测γ-H2AX（DNA双链断裂标志物）的蛋白表达水平；或通过免疫荧光染色观察γ-H2AX焦点形成情况[1]
- MTH1依赖性功能验证：采用siRNA转染技术敲低A549、PC9细胞中的MTH1表达，或通过质粒转染过表达MTH1，随后加入(S)-crizotinib 处理，检测细胞增殖和凋亡率，对比MTH1表达水平对药物效应的影响[2]
- ROS检测：A549细胞经(S)-crizotinib 处理后，加入ROS特异性荧光探针孵育，流式细胞仪检测荧光强度，评估药物对细胞内ROS水平的影响[2]

In six-well plates, cells are plated one day prior to treatment and incubated for 24 hours. The cells were then incubated at 37 °C with 5% CO2 for 7–10 days. The following day, DMSO (equivalent to the highest amount of compound dilution, maximum 0.2%) or compounds were added. Crystal violet solution (0.5% in 25% methanol) is used to stain cells after they have been washed with PBS. Cells are then allowed to dry overnight before being fixed with ice-cold methanol. Crystal violet's ultraviolet absorbance is measured at 595 nm for results quantification after being solubilized in 70% ethanol. GraphPad Prism software is used to analyze data using nonlinear regression.

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Nude mouse xenograft tumor model: 6-8 week-old nude mice were subcutaneously inoculated with log-phase cancer cells (A549 or HCT116, 5×10^6 cells per mouse) on the right back. Seven days after inoculation, when the tumor volume reached approximately 100 mm³, mice were randomly divided into a control group and a treatment group (6 mice per group) [1]
- Administration protocol: (S)-crizotinib was dissolved in normal saline containing 0.5% Tween 80. The treatment group was given oral administration at 50 mg/kg once daily, and the control group was given an equal volume of vehicle for 21 consecutive days [1]
- Monitoring and sample collection: During the experiment, tumor volume (length × width²/2) and mouse body weight were measured every 3 days. After the end of administration, mice were sacrificed, tumor tissues were stripped, weighed and fixed for immunohistochemistry (Ki67, γ-H2AX) and TUNEL apoptosis detection [1]

Absorption
In patients with pancreatic, colorectal, sarcoma, anaplastic large-cell lymphoma and non-small cell lung cancer (NSCLC) treated with crizotinib doses ranging from 100 mg once a day to 300 mg twice a day, the mean AUC and Cmax increased in a dose-proportional manner. A single crizotinib dose of crizotinib is absorbed with a median tmax 4 to 6 hours. In patients receiving multiple doses of crizotinib 250 mg twice daily (n=167), the mean AUC was is 2321.00 ng⋅hr/mL, the mean Cmax was 99.60 ng/mL, and the median tmax was 5.0 hours. The mean absolute bioavailability of crizotinib is 43%, ranging from 32% to 66%. High-fat meals reduce the AUC0-INF and Cmax of crizotinib by approximately 14%. Age, sex at birth, and ethnicity (Asian vs non-Asian patients) did not have a clinically significant effect on crizotinib pharmacokinetics. In patients less than 18 years old, higher body weight was associated with a lower crizotinib exposure.
Route of Elimination
After administering a single 250 mg radiolabeled crizotinib dose to healthy subjects, 63% and 22% of the administered dose were recovered in feces and urine. Unchanged crizotinib represented approximately 53% and 2.3% of the administered dose in feces and urine, respectively.
Volume of Distribution
Following a single intravenous dose, the mean volume of distribution (Vss) of crizotinib was 1772 L.
Clearance
At steady-state (250 mg twice daily), crizotinib has a mean apparent clearance (CL/F) of 60 L/hr. This value is lower than the one detected after a single 250 mg oral dose (100 L/hr),, possibly due to CYP3A auto-inhibition.

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Metabolism / Metabolites
Crizotinib is mainly metabolized in the liver by CYP3A4 and CYP3A5, and undergoes an O-dealkylation, with subsequent phase 2 conjugation. Non-metabolic elimination, such as biliary excretion, can not be excluded. PF-06260182 (with two constituent diastereomers, PF-06270079 and PF-06270080) is the only active metabolite of crizotinib that has been identified. _In vitro_ studies suggest that, compared to crizotinib, PF-06270079 and PF-06270080 are approximately 3- to 8-fold less potent against anaplastic lymphoma kinase (ALK) and 2.5- to 4-fold less potent against Hepatocyte Growth Factor Receptor (HGFR, c-Met).

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Biological Half-Life
Following single doses of crizotinib, the plasma terminal half-life was 42 hours.

Hepatotoxicity
In large early clinical trials, elevations in serum aminotransferase levels occurred in up to 57% of patients treated with standard doses of crizotinib, were greater than 5 times ULN in 6% of patients, and led to early discontinuation of therapy in 2% to 4% of patients. Serum aminotransferase elevations typically arose after 4 to 12 weeks of treatment, but usually without jaundice or alkaline phosphatase elevations. Restarting crizotinib after resolution of the aminotransferase abnormalities can be done starting with a reduced dose. Most cases of liver injury due to crizotinib have been minimally or not symptomatic, and the injury resolved within 1 to 2 months of stopping the drug (Case 1). However, cases with jaundice and symptoms during crizotinib therapy have been reported which were fatal in 0.1% of treated patients (Case 2). The severe cases of liver injury due to crizotinib typically arose within 2 to 6 weeks of starting therapy and presented with marked elevations in serum aminotransferase levels followed by jaundice, progressive hepatic dysfunction, coagulopathy, encephalopathy and death. For these reasons, routine periodic monitoring of liver tests at 2 to 4 week intervals during therapy is recommended. Likelihood score: C (probable cause of clinically apparent acute liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of crizotinib during breastfeeding. Because crizotinib is 91% bound to plasma proteins, the amount in milk is likely to be low. However, its half-life is about 42 hours and it might accumulate in the infant. The manufacturer recommends that breastfeeding be discontinued during crizotinib therapy and for 45 days after the last dose.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Crizotinib is 91% bound to plasma protein. _In vitro_ studies suggest that this is not affected by drug concentration.

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In in vivo experiments, oral administration of (S)-crizotinib at 50 mg/kg for 21 days did not cause significant weight loss in nude mice (weight change rate ≤5%), and no obvious acute toxic reactions such as diarrhea or alopecia were observed [1]
- Serum biochemical tests showed that there were no significant differences in ALT, AST, creatinine, and urea nitrogen levels between the treatment group and the control group, indicating that the drug had no obvious acute damage to liver and kidney functions [1]

[1]. Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy. Nature. 2014 Apr 10;508(7495):222-7.

[2]. (S)-crizotinib induces apoptosis in human non-small cell lung cancer cells by activating ROS independent of MTH1. J Exp Clin Cancer Res. 2017 Sep 7;36(1):120.

Ent-crizotinib is a 3-[1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-[1-(piperidin-4-yl)pyrazol-4-yl]pyridin-2-amine that is the (S)-enantiomer of crizotinib. It is an enantiomer of a crizotinib.

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(S)-crizotinib is the S-enantiomer of crizotinib, a clinically approved ALK/ROS1 inhibitor. (S)-crizotinib has significantly higher affinity for MTH1 than the R-enantiomer (IC50 of R-crizotinib for MTH1=37 nM) and does not inhibit ALK/ROS1 kinase activity [1]
- One of the antitumor mechanisms of (S)-crizotinib is to inhibit MTH1 from scavenging intracellular oxidized nucleotides, reducing the incorporation of erroneous nucleotides into DNA, thereby inducing DNA damage and apoptosis in cancer cells [1]
- In NSCLC cells, the apoptosis-inducing effect of (S)-crizotinib is independent of MTH1 and ROS, and its mechanism is related to downregulating the anti-apoptotic protein Bcl-2, upregulating the pro-apoptotic protein Bax, and activating the mitochondrial apoptotic pathway [2]
- (S)-crizotinib has inhibitory activity against both EGFR-mutant (PC9) and wild-type (A549) NSCLC cells, providing a new potential strategy for lung cancer treatment, especially for ALK/ROS1-negative patients [2]

C21H22CL2FN5O

450.34

449.119

C, 56.01; H, 4.92; Cl, 15.74; F, 4.22; N, 15.55; O, 3.55

1374356-45-2

56671814

Light yellow to yellow solid powder

5.947

77.99

2

6

5

30

558

1

ClC1=C(C([H])=C([H])C(=C1[C@]([H])(C([H])([H])[H])OC1=C(N([H])[H])N=C([H])C(=C1[H])C1C([H])=NN(C=1[H])C1([H])C([H])([H])C([H])([H])N([H])C([H])([H])C1([H])[H])Cl)F

KTEIFNKAUNYNJU-LBPRGKRZSA-N

InChI=1S/C21H22Cl2FN5O/c1-12(19-16(22)2-3-17(24)20(19)23)30-18-8-13(9-27-21(18)25)14-10-28-29(11-14)15-4-6-26-7-5-15/h2-3,8-12,15,26H,4-7H2,1H3,(H2,25,27)/t12-/m0/s1

3-[(1S)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine

S-Crizotinib; PF-2341066; PF2341066; PF02341066; PF-02341066; PF 2341066

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~42 mg/mL (~93.3 mM)
Water: <1 mg/mL
Ethanol: ~22 mg/mL (~48.9 mM)

配方 1 中的溶解度: ≥ 1.25 mg/mL (2.78 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 12.5 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 1.25 mg/mL (2.78 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 12.5 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 1.25 mg/mL (2.78 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 12.5 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。