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

对于 HER2 阴性细胞系（BT474、SK-OV-3），帕罗替尼相当有效，而对于 MDA-MB-231 其抑制作用则不太明显。它阻止 BT474 和 SK-OV 的作用。 -3Pyrotinib 细胞的 IC50 值分别为 5.1 和 43 nM[1]。

吡咯替尼在裸鼠、沉积物和狗中的有效生物利用度分别为 20.6%、43.5% 和 13.5%。剂量分别为5 mg/kg、10 mg/kg和20 mg/kg时，第21天吡咯替尼的TGI%（生长抑制）为109%、157%和159%。在卵巢异种移植模型 SK-OV-3 中，吡咯替尼在 2.5 mg/kg、5 mg/kg 和 10 mg/kg 剂量下，第 21 天的 TGI% 分别为 2%、12% 和 83%。这一额外证据支持吡咯替尼在 10 mg/kg 剂量下具有强大的体内抗肿瘤功效 [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)

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Preclinical toxicity and toxicokinetic (TK) studies[1]
Prior to chronic toxicity studies on rat and dog, dose escalations were investigated in both rats and dogs to determine approximate maximum tolerated dose (MTD), respectively. Then, a rat chronic toxicity study (5, 20, 100 mg/kg) up to 182 days was conducted, followed by a dog chronic toxicity study (3, 10, 30/45 mg/kg) up to 272 days. The corresponding TK and accumulation factor data as well as major findings are discussed in preclinical toxicity section. It should be noted that the highest dose was changed from 45 mg/kg to 30 mg/kg after 15 days due to observed adverse effect during the dog chronic toxicity studies.

[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 is under investigation in clinical trial NCT03756064 (Neoadjuvant Study of Pyrotinib in Patients With HER2 Positive Breast Cancer).
Pyrotinib is an orally bioavailable, dual kinase inhibitor of the epidermal growth factor receptor (EGFR 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.

C32H31CLN6O3

583.079945802689

582.214

C, 65.92; H, 5.36; Cl, 6.08; N, 14.41; O, 8.23

1269662-73-8

(Rac)-Pyrotinib;1246089-97-3;Pyrotinib dimaleate;1397922-61-0

51039030

Typically exists as light yellow to yellow solids at room temperature

5.4

112Ų

2

8

10

42

960

1

CCOC1=C(C=C2C(=C1)N=CC(=C2NC3=CC(=C(C=C3)OCC4=CC=CC=N4)Cl)C#N)NC(=O)/C=C/[C@H]5CCCN5C

SADXACCFNXBCFY-IYNHSRRRSA-N

InChI=1S/C32H31ClN6O3/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)42-20-23-7-4-5-13-35-23/h4-5,7,9-13,15-17,19,24H,3,6,8,14,20H2,1-2H3,(H,36,37)(H,38,40)/b12-10+/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

SHR-1258; SHR1258; SHR-1258; CJN36EQM0H; (R,E)-N-(4-((3-chloro-4-(pyridin-2-ylmethoxy)phenyl)amino)-3-cyano-7-ethoxyquinolin-6-yl)-3-(1-methylpyrrolidin-2-yl)acrylamide; SHR1258; 1269662-73-8 (free base); 2-Propenamide, N-(4-((3-chloro-4-(2-pyridinylmethoxy)phenyl)amino)-3-cyano-7-ethoxy-6-quinolinyl)-3-((2R)-1-methyl-2-pyrrolidinyl)-, (2E)-; SHR 1258

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 : ~10 mg/mL (~17.15 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。