(S)-crizotinib

PTEN (IC50 = 330 nM); NUDIX1

(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]

(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]

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。

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。

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.

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.

[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.

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。