Brigatinib (AP26113)   中文名称: 布格替尼

ALK (IC50 = 0.37 nM); ROS1 (IC50 = 1.9 nM); FLT3 (IC50 = 2.1 nM); IGF1R (IC50 = 24.9 nM); EGFR(C797S/del19) (IC50 = 39.9 nM)

体外活性：AP26113 对敏感和耐药 H3122 细胞均具有高度活性，以剂量依赖性方式减少细胞生长、抑制 ALK 磷酸化并诱导细胞凋亡。 AP26113 降低 H3122 和 H3122 CR 细胞中的 p-ALK，IC50 值分别为 7.4 和 16.8 nM。 AP26113 减少表达天然或突变 EML4-ALK 的 Ba/F3 细胞中的细胞数量，IC50 分别为 10 nM 和 24 nM。 AP26113 抑制 SU-DHL-1、H3122 和 Ba/F3-EML4-ALK v1 细胞系的细胞生长，GI50 分别为 9 nM、4 nM 和 13 nM。 AP26113 在 Karpas-299、SU-DHL-1 和 L-82 细胞系中抑制 ALK 磷酸化，IC50 分别为 3.2 nM、1.5 nM 和 2.1 nM。 AP26113 剂量依赖性地抑制 Karpas-299 和 H3122 细胞中 ALK 和 ERK 的磷酸化。 AP26113 抑制 Ba/F3 系（天然 EML4-ALK）和 Ba/F3 系（EML4-ALK G1269S 突变体）中的细胞生长，IC50 分别为 11 nM 和 16 nM。 AP26113 抑制 Ba/F3 系（天然 EML4-ALK）和 Ba/F3 系（EML4-ALK E1210K 突变体）中的 ALK 磷酸化，IC50 分别为 74 nM 和 335 nM。 AP26113 (10 mg/kg-75 mg/kg) 在 PF-02341066 耐药 EML4-ALK 突变小鼠异种移植模型中有效。 AP26113 分别以 25 mg/kg、50 mg/kg 和 50 mg/kg 剂量诱导表达天然 EML4-ALK 以及 G1269S 和 L1196M 突变体的肿瘤消退。在表达 EGFR-DEL 的 Ba/F3 细胞中，AP26113 抑制 EGFR 磷酸化和活力，IC50 分别为 75 nM 和 114 nM。在表达 EGFR-DEL/T790M 的 Ba/F3 细胞中，AP26113 抑制 EGFR 磷酸化和活力，IC50 分别为 15 和 281 nM。 AP26113 在表达 EGFR-DEL (HCC827) 的 NSCLC 系中抑制 EGFR 磷酸化，IC50 为 62 nM，抑制细胞生长，GI50 为 165 nM。 AP26113 在表达 EGFR-DEL/T790M 的 HCC827 细胞中抑制 EGFR 磷酸化，IC50 为 59 nM，抑制细胞生长，GI50 为 245 nM。 AP26113 在 HCC78 NSCLC 细胞中以剂量依赖性方式有效抑制 SLC34A2-ROS 驱动的信号传导和增殖。激酶测定：对 289 种激酶进行体外 HotSpotSM 激酶分析。该测定在 10 μM [33P]-ATP 存在下进行，使用 brigatinib 浓度范围为 0.05 nM 至 1 μM。细胞测定：使用 Cell Titer 96 Aqueous One Solution 细胞增殖测定或 CyQuant 细胞增殖测定评估细胞生长。铺板后 24 小时，用 AP26113 处理细胞并生长 72 小时。引起 50% 生长抑制 (GI50) 的浓度通过校正零时间（处理时间）的细胞计数并使用 XLfit 版本 4.2.2 for Microsoft Excel 将数据绘制为相对于媒介物 (DMSO) 处理的细胞的生长百分比来确定。使用 SU-DHL-1、H3122 和 Ba/F3-EML4-ALK v1 细胞系。

AP26113 (< 50 mg/kg) 剂量依赖性地抑制 Karpas-299 异种移植小鼠模型肿瘤中的 p-ALK。 AP26113 (< 50 mg/kg) 剂量依赖性地抑制 Karpas-299 异种移植小鼠模型和 H3122 异种移植小鼠模型中的肿瘤生长。 AP26113 表现出良好的特性，包括中等的体外血浆蛋白结合（在人、大鼠、小鼠中为 47%、70% 和 76%），对主要 CYP 亚型的抑制可忽略不计。 AP26113 (10 mg/kg) 在大鼠中具有良好的耐受性，Cmax 为 2587 ng/mL，AUC 为 41120 hr.ng/mL。 AP26113 (25 mg/kg) 导致 HCC827(EGFR-DEL) 或 HCC827(EGFR-DEL/T790M) 异种移植小鼠模型中的肿瘤消退。

在体外对 289 种激酶进行 HotSpot^SM 激酶谱分析。该实验在 10 μM [³³P]-ATP 存在的情况下，使用浓度范围为 0.05 nM 至 1 μM 的布加替尼进行。

将指定的抑制剂连续稀释并添加到含有 15,000 个细胞的每个孔中。刃天青在 72 小时后测量细胞的活力。通过将数据拟合到对数（抑制剂浓度）与标准化响应（可变斜率）的方程中，使用 GraphPad Prism 6.0 确定 IC50 值。每个实验均进行两份，至少进行三次。

Mice: (1) Female SCID/beige mice, aged eight to ten weeks, receive intravenous injections of 5x10⁶ H3122 cells each. After the tumor size reaches approximately 300 mm³ on day zero, the mice are randomized into ten treatment groups. Treatments are taken orally at a dose volume of 10 mL/kg for a maximum of 21 days in a row. Tumors under the skin are measured twice or three times a week. The formula (L×W²)/2 is used to calculate the tumor volume (in mm³). The animal is put to sleep by CO₂ asphyxiation when a tumor weighs 10% of its body weight. (2) Female SCID/beige mice, aged eight to ten weeks, receive subcutaneous injections of 2.5 ×10⁶ Karpas-299 cells per mouse. After the tumors reach approximately 180 mm³ on day zero, the mice are randomly assigned to one of ten treatment groups. Oral treatment is given for 14 days in a row at a dose volume of 10 mL/kg. The measurement and computation of tumor volume follow the guidelines for the H3122 model.

Absorption, Distribution and Excretion
Administration of brigatinib at a concentration of 90 mg generates a Cmax of 552 ng/ml and AUC of 8165 ng h/ml while the administration of 180 mg presents a Cmax of 1452 ng/ml and AUC of 20276 ng h/ml. It has a dose proportional exposure with an accumulation ratio on the range of 1.9 to 2.4. Following oral administration of brigatinib, the Tmax is presented in a range from 1 to 4 hours. Consumption of a high-fat meal compared to overnight fasting reduces Cmax by 13% without presenting an effect on AUC.
The elimination of brigatinib is divided in 65% in feces and 25% in urine. From the elimination in both compartments, the unchanged for of brigatinib represented 41% of the total in feces and 86% in urine.
The apparent volume of distribution at steady state is 153 L.
After oral administration of180 mg of brigatinib, the apparent oral clearance at steady-state is 12.7 L/h.

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Metabolism / Metabolites
Brigatinib is metabolized by CYP2C8 (72.4%) and CYP3A4 (27.6%) in human liver microsomes and hepatocytes. The two major metabolites generated are the N-demethylated form and the cysteine conjugated form. Oral administration of radiolabelled brigatinib showed the systemic presence of 91.5% in the unchanged form and 3.5% of the primary metabolite AP26123. The AUC of AP26123 is less than 10% of the AUC of brigatinib and presented an inhibitory effect 3 fold lower.

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Biological Half-Life
The half-life of brigatinib at steady-state was 25 hours.

Hepatotoxicity
In preregistration trials of brigatinib, ALT elevations occurred in up to 40% of patients but values above 5 times the upper limit of normal (ULN) were found in only 1% to 3%. Brigatinib therapy was also associated with frequent elevations in alkaline phosphatase (15% to 29%), but the serum enzyme elevations were usually mild-to-moderate in degree as well as asymptomatic and transient in nature. Clinically apparent liver injury with jaundice was not reported in the prelicensure studies of brigatinib and no reports have been published since its approval. In general, the ALK kinase inhibitors are associated with a high rate of serum enzymes elevations during therapy, but convincing cases of idiosyncratic, clinically apparent liver injury from their use have been rare. Most cases have been reported with crizotinib [approved in 2011] which is also the most frequently used of the kinase inhibitors with activity against ALK. Cases of liver injury with jaundice were reported to occur in trials of alectinib [2015] and ceritinib [2014], but details were not provided. Thus, acute liver injury with jaundice may occur with brigatinib but it must be rare if it occurs at all.
Likelihood score: E* (unproved but suspected 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 brigatinib during breastfeeding. The manufacturer recommends that breastfeeding be discontinued during brigatinib therapy and for 1 week after the final 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
66% of brigatinib dose is bound to plasma proteins, which gives a blood-to-plasma concentration ratio of 0.69.

[1]. The Potent ALK Inhibitor Brigatinib (AP26113) Overcomes Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in Preclinical Models. Clin Cancer Res. 2016 Nov 15;22(22):5527-5538.

[2]. Discovery of Brigatinib (AP26113), a Phosphine Oxide-Containing, Potent, Orally Active Inhibitor of Anaplastic Lymphoma Kinase. J Med Chem. 2016 May 26;59(10):4948-64.

[3]. Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK-positive neuroblastoma cells, Drosophila and mice. Oncotarget. 2016 May 17;7(20):29011-22.

Brigatinib, originally named AP26113, is a reversible dual inhibitor of anaplastic lymphoma kinase (ALK) and epidermal growth factor receptor (EGFR). It presents selectivity against the mutant forms of EGFR compared to the wild-type. It also exhibits selectivity against 9 different Crizotinib-resistant mutants of the EML4-ALK fusion gene, which is a pivotal player in the transformation of susceptible lung parenchyma. Brigatinib was developed by Ariad Pharmaceuticals, a subsidiary of Takeda Pharmaceutical Company Limited, and FDA-approved on April 28, 2017.
Brigatinib is a Kinase Inhibitor. The mechanism of action of brigatinib is as a Tyrosine Kinase Inhibitor, and Cytochrome P450 3A Inducer.
Brigatinib is a tyrosine kinase receptor inhibitor and antineoplastic agent used in the therapy of selected forms of advanced non-small cell lung cancer. Brigatinib is associated with a moderate rate of transient elevations in serum aminotransferase levels during therapy but has yet to be linked to instances of clinically apparent acute liver injury.
Brigatinib is an orally available inhibitor of receptor tyrosine kinases anaplastic lymphoma kinase (ALK) and the epidermal growth factor receptor (EGFR) with potential antineoplastic activity. Brigatinib binds to and inhibits ALK kinase and ALK fusion proteins as well as EGFR and mutant forms. This leads to the inhibition of ALK kinase and EGFR kinase, disrupts their signaling pathways and eventually inhibits tumor cell growth in susceptible tumor cells. In addition, AP26113 appears to overcome mutation-based resistance. ALK belongs to the insulin receptor superfamily and plays an important role in nervous system development; ALK dysregulation and gene rearrangements are associated with a series of tumors. EGFR is overexpressed in a variety of cancer cell types.

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Drug Indication
The anaplastic lymphoma kinase positive, metastatic non-small cell lung cancer (ALK+ NSCLC), represents only 3-5% of the NSCLC cancer cases, but the ALK mutation, overexpression and presence in several oncogenic fusion proteins in solid and hematologic tumors have pointed out the importance as well as its potential as a cancer therapy target. The ALK-related cases of NSCLC are associated with the presence of the fusion gene EML4-ALK which fused the ALK protein with the echinoderm microtubule-associated protein like-4 whose original function is the correct formation of microtubules. The presence of the aberrant fusion protein results in abnormal signaling that provokes increased cell growth, proliferation and survival. Crizotinib is indicated for the treatment of such cases but the presence of ALK kinase domain mutations confer resistance to the treatment. Thus, brigatinib is indicated for the treatment of patients with ALK+ NSCLC with intolerance to Crizotinib.
FDA Label
Alunbrig is indicated as monotherapy for the treatment of adult patients with anaplastic lymphoma kinase (ALK)‑positive advanced non‑small cell lung cancer (NSCLC) previously not treated with an ALK inhibitor. Alunbrig is indicated as monotherapy for the treatment of adult patients with anaplastic lymphoma kinase ALKpositive advanced NSCLC previously treated with crizotinib.
Treatment of anaplastic large cell lymphoma, Treatment of inflammatory myofibroblastic tumours, Treatment of non-small cell lung cancer

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Mechanism of Action
Brigitanib acts as a tyrosine kinase inhibitor with activity against multiple kinases including ALK, ROS1, insulin-like growth factor 1 receptor and against EGFR deletions and point mutations. It acts by inhibiting ALK phosphorylation and the activation of downstream signaling proteins.

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Pharmacodynamics
Brigitanib inhibits proliferation and in vitro viability of cells expressing the fusion protein EML4-ALK as well as 17 crizotinib-resistant ALK mutants. Its action is expanded to cells expressing EGFR deletions, ROS1-L2026M, FLT3-F691L and FLT3-D835Y. Brigitanib presents a dose-dependent inhibition of tumor growth, tumor burden and prolonged survival in mice EML4-ALK xenograft models. Time course of Brigatinib and exposure-response studies are still unknown.

C₂₉H₃₉CLN₇O₂P

584.09

583.259

C, 59.63; H, 6.73; Cl, 6.07; N, 16.79; O, 5.48; P, 5.30

1197953-54-0

Brigatinib-13C6

68165256

Light yellow solid powder

1.3±0.1 g/cm3

781.8±70.0 °C at 760 mmHg

426.6±35.7 °C

0.0±2.7 mmHg at 25°C

1.641

0.43

95.67

2

9

8

40

835

0

ClC1=C([H])N=C(N=C1N([H])C1=C([H])C([H])=C([H])C([H])=C1P(C([H])([H])[H])(C([H])([H])[H])=O)N([H])C1C([H])=C([H])C(=C([H])C=1OC([H])([H])[H])N1C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N1C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])C1([H])[H]

AILRADAXUVEEIR-UHFFFAOYSA-N

InChI=1S/C29H39ClN7O2P/c1-35-15-17-37(18-16-35)21-11-13-36(14-12-21)22-9-10-24(26(19-22)39-2)33-29-31-20-23(30)28(34-29)32-25-7-5-6-8-27(25)40(3,4)38/h5-10,19-21H,11-18H2,1-4H3,(H2,31,32,33,34)

5-chloro-4-N-(2-dimethylphosphorylphenyl)-2-N-[2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl]pyrimidine-2,4-diamine

AP-26113; AP 26113; Brigatinib-analog; AP26113; Brigatinib; Alunbrig.

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)

配方 1 中的溶解度: ≥ 1 mg/mL (1.71 mM) (饱和度未知) in 10% EtOH + 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，将 100 μL 10.0 mg/mL 澄清乙醇储备液加入到 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: ≥ 1 mg/mL (1.71 mM) (饱和度未知) in 10% EtOH + 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 10.0 mg/mL 澄清乙醇储备液加入 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 mg/mL (1.71 mM) (饱和度未知) in 10% EtOH + 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 10.0 mg/mL 澄清 EtOH 储备液添加到 900 μL 玉米油中并充分混合。

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配方 4 中的溶解度: ≥ 0.5 mg/mL (0.86 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，将100 μL 5.0 mg/mL澄清DMSO储备液加入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 5 中的溶解度: ≥ 0.5 mg/mL (0.86 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，将 100 μL 5.0 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 6 中的溶解度: ≥ 0.5 mg/mL (0.86 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 5.0 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 7 中的溶解度: NMP+polyethylene glycol 300 (10+90, v+v): 1 mg/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;
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