ALK^L1196 (IC50 = 15-43 nM); ALK^G1269A (IC50 = 14-80 nM); ALK^1151Tins (IC50 = 38-50 nM); ALK^G1202R (IC50 = 77-113 nM); ALK^WT (IC50 <0.07 nM); ALK^L1996M (IC50 = 0.6 nM); ALK^G1269A (IC50 = 0.9 nM); ALK^1151Tins (IC50 = 0.1 nM); ALK^L1152R (IC50 <0.1 nM); ALK^S1206Y (IC50 = 0.2 nM); ALK^C1156Y (IC50 <0.1 nM); ALK^F1174L (IC50 <0.1nM)

体外活性：PF-06463922 表现出针对 ALK 和大量 ALK 临床突变的显着细胞活性，IC50 范围为 0.2 nM-77 nM。PF-06463922 显着抑制含有 SLC34A2 的 HCC78 人 NSCLC 细胞的细胞增殖并诱导细胞凋亡-ROS1融合和表达人CD74-ROS1的BaF3-CD74-ROS1细胞。 PF-06463922 还显示出有效的生长抑制活性，并诱导含有非突变 ALK 或突变 ALK 融合的 NSCLC 细胞凋亡激酶测定：重组人野生型和突变 ALK 激酶结构域蛋白（氨基酸 1093-1411）产生于-使用杆状病毒表达，通过 MgATP 自磷酸化预激活，并使用微流体迁移率变化测定法测定激酶活性。反应含有 1.3 nM 野生型 ALK 或 0.5 nM 突变型 ALK（适合反应 1 小时后肽底物产生 15-20% 磷酸化）、3 μM 5-FAM-KKSRGDYMTMQIG-CONH2）、5 mM MgCl2 和25 mM Hepes（pH 7.1）中的 ATP 浓度（Km）。动力学和晶体学研究显示该抑制剂具有 ATP 竞争性。 Ki 值是通过将转化率 (%) 拟合到竞争性抑制方程来计算的。 ROS1 酶的测定方法如上所述，但使用 0.25 nM 重组人 ROS1 催化结构域（氨基酸 1883-2347）。使用 206 激酶面板评估激酶抑制剂选择性。细胞测定：将细胞接种到 96 孔板中含有 10% FBS 的生长培养基中，并在 37°C 下培养过夜。第二天，将连续稀释的 Lorlatinib 或适当的对照添加到指定孔中，并将细胞在 37°C 下孵育 72 小时。进行 CellTiter-Glo 测定以确定相对细胞数。 IC 50 值通过使用四参数分析方法的浓度-响应曲线拟合来计算。

如前所述，LSL-FIG-ROS1；Cdkn2a−/−；LSL-Luc 小鼠通过颅内立体定向注射 Adeno-Cre 启动 GBM 肿瘤的从头发生。如下所述，使用 BLI 监测肿瘤发展。一旦肿瘤达到给定大小（107p-1·s-1·cm-2·sr-1），将动物随机纳入载体对照或用指定剂量的Lorlatinib进行3、7或14天治疗。药物通过皮下植入的 Alzet 渗透泵给药。治疗后，处死小鼠，对 GBM 肿瘤进行显微解剖，并将组织在液氮中快速冷冻。剩余的大脑进行组织学处理。 在大鼠中，PF-06463922 显示出低血浆清除率、中等分布体积、合理的半衰期、p-糖蛋白 1 介导的外排倾向低以及 100% 的生物利用度。在体内，PF-06463922 通过抑制 ROS1 磷酸化和下游信号分子，以及抑制肿瘤中的细胞周期蛋白 Cyclin D1，在表达人 CD74-ROS1 和 Fig-ROS1 的 NIH3T3 异种移植模型中显示出细胞减灭性抗肿瘤功效。在体内，PF-06463922还在携带表达EML4-ALK、EML4-ALK-L1196M、EML4-ALK-G1269A、EML4-ALK-G1202R或NPM-ALK的肿瘤异种移植物的小鼠中表现出显着的抗肿瘤活性。

微流体迁移率变动测定用于测量重组人野生型和突变型 ALK 激酶结构域蛋白（氨基酸 1093-1411）中的激酶活性，这些蛋白是通过杆状病毒表达和 MgATP 自磷酸化内部产生的。反应含有 3 μM 5-FAM-KKSRGDYMTMQIG-CONH2)、5 mM MgCl2、1.3 nM 野生型 ALK 或 0.5 nM 突变型 ALK（适合在反应 1 小时后产生 15-20% 的肽底物磷酸化），以及25 mM Hepes（pH 7.1）中的 ATP 浓度（Km）。动力学和晶体学研究的结果表明该抑制剂具有 ATP 竞争性。将转化率 (%) 拟合到竞争性抑制方程即可得出 Ki 值。检测 ROS1 酶的程序与检测 ALK 的程序相同，不同之处在于使用 0.25 nM 重组人 ROS1 催化结构域（氨基酸 1883-2347）。 206 激酶组用于评估激酶抑制剂的选择性。

在 96 孔板中，将细胞接种在含有 10% FBS 的生长培养基中，并在 37°C 下孵育整晚。第二天将连续稀释的 Lorlatinib 或合适的对照添加到指定的孔中后，将细胞在 37°C 下孵育 72 小时。为了确定相对细胞数，进行了 CellTiter-Glo 测定。使用四参数分析方法来拟合浓度-响应曲线并确定IC50值。

In LSL-FIG-ROS1;Cdkn2a−/−;LSL-Luc mice, de novoGBM tumorigenesis is induced by intracranial stereotactic injections of Adeno-Cre, as previously reported. BLI is used to track the development of tumors as will be discussed below. Animals are randomly assigned to either vehicle control or 3-, 7-, or 14-day treatments with the prescribed doses of lerlatinib once tumors reach a specific size (107 p ^-1·s ^-1·cm ^-2·sr ^-1). The medication is delivered via s.c. implanted Alzet osmotic pumps. Following therapy, GBM tumors are microdissected, tissues are flash-frozen in liquid N2, and mice are killed. For histology, the remaining brains are processed.

Absorption, Distribution and Excretion
The median lorlatinib Tmax was 1.2 hours (0.5 to 4 hours) following a single oral 100 mg dose and 2 hours (0.5 to 23 hours) following 100 mg orally once daily at steady state. The mean absolute bioavailability is 81% (90% CI 75.7%, 86.2%) after oral administration compared to intravenous administration. Administration of lorlatinib with a high fat, high-calorie meal (approximately 1000 calories with 150 calories from protein, 250 calories from carbohydrate, and 500 to 600 calories from fat) had no clinically meaningful effect on lorlatinib pharmacokinetics.
Following a single oral 100 mg dose of radiolabeled lorlatinib, 48% of the radioactivity was recovered in urine (<1% as unchanged) and 41% in feces (about 9% as unchanged).
The mean (CV%) steady-state volume of distribution (Vss) was 305 L (28%) following a single intravenous dose.
The mean oral clearance (CL/F) was 11 L/h (35%) following a single oral 100 mg dose and increased to 18 L/h (39%) at steady state, suggesting autoinduction.

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Metabolism / Metabolites
In vitro, lorlatinib is metabolized primarily by CYP3A4 and UGT1A4, with minor contribution from CYP2C8, CYP2C19, CYP3A5, and UGT1A3. In plasma, a benzoic acid metabolite (M8) of lorlatinib resulting from the oxidative cleavage of the amide and aromatic ether bonds of lorlatinib accounted for 21% of the circulating radioactivity in a human [14C] mass balance study. The oxidative cleavage metabolite, M8, is pharmacologically inactive.

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Biological Half-Life
The mean plasma half-life (t½) of lorlatinib was 24 hours (40%) after a single oral 100 mg dose of lorlatinib.

Hepatotoxicity
In large early clinical trials, elevations in serum aminotransferase levels occurred in up to 28% of patients treated with standard doses of lorlatinib but were above 5 times ULN in only 2% of patients and rarely led to early discontinuation of therapy. The abnormalities were typically transient, asymptomatic and not associated with jaundice. In prelicensure clinical trials there were no instances of clinically apparent liver injury. In contrast, most other ALK inhibitors have been linked to instances of acute liver injury which can be severe and even fatal. The liver injury typically arises within 4 to 12 weeks of starting therapy and presents with marked elevations in serum aminotransferase levels followed by jaundice and progressive hepatic dysfunction. While lorlatinib has not been associated with instances of severe liver injury, it has had limited clinical use.
Likelihood score: E* (unproven but suspected rare cause of clinically apparent 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 lorlatinib during breastfeeding. The manufacturer recommends that breastfeeding be discontinued during lorlatinib therapy and for 7 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
In vitro, lorlatinib was 66% bound to plasma proteins at a concentration of 2.4 µM. The blood-to-plasma ratio was 0.99.

[1]. J Med Chem . 2014 Jun 12;57(11):4720-44.

[2].Mol Cancer Ther (2013) 12 (11_Supplement): A277.

[3]. Mol Cancer Ther (2013) 12 (11_Supplement): C253.

Pharmacodynamics
Based on data from Study B7461001, exposure-response relationships for Grade 3 or 4 hypercholesterolemia and for any Grade 3 or 4 adverse reaction were observed at steady-state exposures achieved at the recommended dosage, with higher probability of the occurrence of adverse reactions with increasing lorlatinib exposure. In 295 patients who received lorlatinib at the recommended dosage of 100 mg once daily and had an ECG measurement in the same Study B7461001, the maximum mean change from baseline for their PR interval was 16.4 ms (2-sided 90% upper confidence interval [CI] 19.4 ms). Among the 284 patients with PR interval <200 ms at baseline, 14% had PR interval prolongation ≥200 ms after starting use with lorlatinib. The prolongation of PR interval occurred in a concentration-dependent manner and atrioventricular block occurred in 1% of patients. Finally, in 275 patients who received lorlatinib at the recommended dosage in the activity-estimating portion of Study B7461001, no large mean increases from baseline in the QTcF interval (i.e., >20 ms) were detected.

C21H19FN6O2

406.41

406.155

C, 62.06; H, 4.71; F, 4.67; N, 20.68; O, 7.87

1454846-35-5

1924207-18-0 (acetate);2135926-03-1;1454846-35-5;2306217-6 (hydrate);

71731823

White to off-white solid powder

1.4±0.1 g/cm3

675.0±55.0 °C at 760 mmHg

362.1±31.5 °C

0.0±2.1 mmHg at 25°C

1.687

1.24

110.06

1

7

0

30

700

1

FC1C([H])=C([H])C2C(N(C([H])([H])[H])C([H])([H])C3C(=C(C#N)N(C([H])([H])[H])N=3)C3C([H])=NC(=C(C=3[H])O[C@]([H])(C([H])([H])[H])C=2C=1[H])N([H])[H])=O

IIXWYSCJSQVBQM-LLVKDONJSA-N

InChI=1S/C21H19FN6O2/c1-11-15-7-13(22)4-5-14(15)21(29)27(2)10-16-19(17(8-23)28(3)26-16)12-6-18(30-11)20(24)25-9-12/h4-7,9,11H,10H2,1-3H3,(H2,24,25)/t11-/m1/s1

(16R)-19-amino-13-fluoro-4,8,16-trimethyl-9-oxo-17-oxa-4,5,8,20-tetrazatetracyclo[16.3.1.02,6.010,15]docosa-1(22),2,5,10(15),11,13,18,20-octaene-3-carbonitrile

Lorbrena; PF06463922; PF-6463922; PF6463922; PF 6463922; PF 06463922; PF-06463922;Lorlatinib; 1454846-35-5; Lorbrena; Lorviqua; lorlatinibum; PF06463922

2934.99.09.01

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: ~81 mg/mL (~199.3 mM)
Water: <1 mg/mL
Ethanol: ~30 mg/mL warmed (~73.8 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.15 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 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 (6.15 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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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.15 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 25.0 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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配方 4 中的溶解度: ≥ 2.5 mg/mL (6.15 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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 5 中的溶解度: 2% DMSO+30% PEG 300+ddH2O: 5mg/mL

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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。