EGFR (IC50 = 13 nM); HER2 (IC50 = 38 nM)

在HER2依赖性细胞系（BT474、SK-OV-3）中，马来酸帕罗替尼表现出很强的抑制作用；在 HER2 阴性细胞系 (MDA-MB-231) 中，效果较差。 BT474 和 SK-OV-3 细胞被吡咯替尼二马来酸盐抑制，IC50 值分别为 5.1 和 43 nM。在包括 KDR、c-Kit、PDGFRβ、c-Src 和 C-Met 在内的一组激酶中进行测试时，马来酸吡咯替尼表现出与 HKI-272 相同的高选择性（c-Src 的 IC50 为 790 nM，其他 >3000纳米）[1]。

马来酸吡咯替尼在大鼠、狗和裸鼠中的耐受生物利用度分别为 20.6%、43.5% 和 13.5%。马来酸吡咯替尼的理化性质与药物相似。它还显示出在人类受试者中相对较高的口服暴露量（口服；t1/2=15小时），其半衰期短于临床前动物物种，例如大鼠（iv；t1/2=1.56小时；ig ；t1/2= 2.52 h）和小鼠（iv；t1/2=4.42 h；ig；t1/2=3.38 h）[1]。

EGFR/HER2激酶抑制试验用于测定化合物的体外活性。通过将一系列浓度的受试化合物与特定酶和底物孵育，测量每种化合物的半最大抑制浓度IC50（受试化合物显示酶活性50%抑制的浓度）。EGFR激酶测定使用了一种人源重组蛋白，该蛋白在含有60 mM HEPES（pH 7.5）、5 mM MgCl2、5 mM MnCl2、3μM Na3VO4、1.25 M DTT和20μM ATP的混合物的缓冲溶液中，在25°C下与肽底物在不同浓度的测试化合物反应45分钟。HER2激酶测定试剂盒与蛋白质底物（Tyr 87）在包含60 mM HEES（pH 7.5通过时间分辨荧光法测定EGFR和HER2激酶活性[1]。

体外细胞增殖抑制测定的一般程序在合适浓度（例如5000个细胞/mL培养基）的癌症细胞（A431、SK-BR-3和NCI-N87）上进行。然后将细胞在二氧化碳（5%CO2）培养箱中孵育，直到它们达到85%的融合，随后，用新鲜的细胞培养基替换细胞培养基，并以一系列浓度（通常为6至7个浓度）添加测试化合物。然后将细胞放回培养箱中并连续培养。72小时后，通过磺基罗丹明B（SRB）法测定测试化合物抑制细胞增殖的活性。IC50值由试验化合物系列浓度的抑制率数据计算[1]。

In vivo efficacy studies were performed on BALB/Ca-nude mice (6 to 7 weeks, female) from SLAC. Nude mice were hypodermic inoculated BT-474 human breast cancer cell or SK-OV-3 ovarian cancer cell. After tumor grew to 150–250 mm3, mice were randomly divided into groups and dosed once daily. The volume of tumors and the weight of the mice were measured and recorded for 2–3 times per week. The volume of tumor (V) was calculated as V = 1/2 xaxb2 (a: length of tumors, b: width of tumors). Tumor growth inhibition (TGI) was calculated as: TGI (%) = 100 − (VT − VT0) / (VC − VC0) ∗ 100%; where VT0 and VT are the tumor volumes of the beginning and finish days of dosed groups, respectively; and VC0 and VC are the tumor volumes of the beginning and finish days for the control group, respectively. In the case of tumor regression, TGI was calculated as: TGI (%) = 100 − (VT − VT0) / VT0 ∗ 100.[1]

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In vivo PK and human PK studies[1] Animals utilized for preclinical studies include nude mice, rats and dogs. All animals were treated in accordance with Institutional Guide for the Care and Use of Laboratory Animals. Nude mice (around 20 g, 9 males and 9 females) were purchased from Sino-British Sippr/BK Lab Animal Co. Ltd. (Shanghai) (SCXK 2013-0016), Sprague Dawley (SD) rats (200–250 g, 3 males and 3 females) from Shanghai SLAC Laboratory Animal Co., LTD (SYXK 2003-0029), and beagle dogs (9–13 kg, 2 males and 2 females) from Beijing Marshall Biotechnology Co., Ltd. (SCXK 2009-0002), respectively. Briefly, test compounds were administrated in both intravenous (i.v.) and intragastric (i.g.) for mice, rats and dogs in order to obtain their bioavailability. Plasma samples of nude mice, SD rats and dogs were collected at pre-dose and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 h after the IV administration, plasma samples of SD rats were collected at pre-dose and 1.0, 2.0, 3.0, 3.5, 4, 4.5, 5, 6, 8, 12, 24 h and plasma samples of beagle dogs were collected at pre-dose and 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24 h after the i.g. dose. Human sample collection was conducted in Teda International Cardiovascular Hospital with study protocol approved by the ethics committee of the hospital. Written informed consent was obtained from the subjects enrolled in this study. Ten healthy subjects participated in the study for human PK and metabolite identification as well as elimination study. After an overnight fast, each subject received a single oral administration of 240 mg pyrotinib maleate tablet. Plasma samples were collected at pre-dose and 0.5, 1, 2, 3, 4, 5, 6, 7, 9, 12, 24, 36, 48, 72, and 96 h post-dose. Urine samples were collected at pre-dose and 0–4, 4–8, 8–12, 12–24, 24–36, 36–48, 48–72, and 72–96 h post-dose. Feces samples were collected at pre-dose and 0–24, 24–48, 48–72, and 72–96 h post-dose. All the samples were preserved at − 80 °C until analysis. A range of five doses (80, 160, 240, 320 and 400 mg) was conducted for Phase I dose escalation study. All PK and TK parameters were calculated throughout a non-compartmental model using Phoenix WinNonlin software (5.2)

[1]. Discovery and development of Pyrotinib: A novel irreversible EGFR/HER2 dual tyrosine kinase inhibitor with favorable safety profiles for the treatment of breast cancer. Eur J Pharm Sci. 2017 Jan 21. pii: S0928-0987(17)30043-X.

Pyrotinib Dimaleate is the dimaleate ester of pyrotinib, an orally bioavailable, dual kinase inhibitor of the epidermal growth factor receptor (EGFR, ErbB1 or HER-1) and the human epidermal growth factor receptor 2 (ErbB2 or HER-2), with potential antineoplastic activity. Upon oral administration, pyrotinib binds to and inhibits both EGFR and HER2, which may result in the inhibition of tumor growth and angiogenesis, and tumor regression in EGFR/HER2-expressing tumor cells. EGFR and HER2 are receptor tyrosine kinases that are upregulated in various tumor cell types and play major roles in tumor cell proliferation and tumor vascularization.

C36H35CLN6O7

815.22

814.236

C, 61.84; H, 5.05; Cl, 5.07; N, 12.02; O, 16.02

1397922-61-0

Pyrotinib;1269662-73-8

72710866

Typically exists as light yellow to yellow solids at room temperature

262Ų

6

16

14

58

1080

1

CCOC1=C(C=C2C(=C(C=NC2=C1)C#N)NC3=CC(=C(C=C3)OCC4=CC=CC=N4)Cl)NC(=O)/C=C/[C@@H]5N(CCC5)C.C(=C\C(=O)O)\C(=O)O.C(=C\C(=O)O)\C(=O)O

ZUZJPRMSUMMELD-ZLWATVBPSA-N

InChI=1S/C32H31ClN6O3.C4H4O4/c1-3-41-30-17-27-25(16-28(30)38-31(40)12-10-24-8-6-14-39(24)2)32(21(18-34)19-36-27)37-22-9-11-29(26(33)15-22)42-20-23-7-4-5-13-35-235-3(6)1-2-4(7)8/h4-5,7,9-13,15-17,19,24H,3,6,8,14,20H2,1-2H3,(H,36,37)(H,38,40)1-2H,(H,5,6)(H,7,8)/b12-10+2-1-/t24-/m1./s1

(R,E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)-3-(1-methylpyrrolidin-2-yl)acrylamide maleate

SHR-1258; SHR1258; SHR-1258 dimaleate; Pyrotinib Maleate; 1397922-61-0; 85KUE857XM; 2-Propenamide, N-(4-((3-chloro-4-(2-pyridinylmethoxy)phenyl)amino)-3-cyano-7-ethoxy-6-quinolinyl)-3-((2R)-1-methyl-2-pyrrolidinyl)-, (2E)-, (2Z)-2-butenedioate (1:2); (Z)-but-2-enedioic acid;(E)-N-[4-[3-chloro-4-(pyridin-2-ylmethoxy)anilino]-3-cyano-7-ethoxyquinolin-6-yl]-3-[(2R)-1-methylpyrrolidin-2-yl]prop-2-enamide; UNII-85KUE857XM; SHR 1258.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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

H2O : ≥ 106 mg/mL (~130.03 mM)
DMSO : ~100 mg/mL (~122.67 mM)

配方 1 中的溶解度: 2.5 mg/mL (3.07 mM) 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；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: 2.5 mg/mL (3.07 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 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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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。