BTK/Bruton tyrosine kinase

在生化和细胞检测中，BGB-3111 对 BTK 与 EGFR、FGR、FRK、HER2、HER4、ITK、JAK3、LCK、BLK 和 TEC 的选择性比依鲁替尼更高[1]。
BGB-3111 有效抑制细胞通过阻断下游 PLC-γ2 信号传导并抑制 BCR 聚集触发的 BTK 自磷酸化来抑制多种 MCL 和 DLBCL 细胞系的增殖 [2]。

在临床前动物研究中，BGB-3111 表现出比依鲁替尼更好的口服生物利用度，在组织中实现了更高的暴露量和更完全的靶标抑制[1]。
BGB-3111 治疗导致小鼠 BTK 占用测定中出现剂量依赖性 BTK 占用，并且它在靶器官（如脾脏和 PBMC）中的效力约为依鲁替尼的三倍[2]。
BGB-3111 比依鲁替尼表现出更好的疗效，并在 REC-1 MCL 中诱导剂量依赖性抗肿瘤作用和 ABC 亚型 DLBCL (TMD-8) 异种移植模型。根据一项大鼠毒性研究，BGB-3111 具有高度良好的耐受性，即使剂量高达 250 mg/kg/天，也未达到 MTD[3]。

蛋白质沉淀实验程序:[4]
将人BTK蛋白与BGB-3111 (31a, Zanubrutinib)或B43(一种不含反应性化学基团的可逆BTK抑制剂)预孵育，形成BTK/化合物复合物(过量的BTK激酶以确保BTK蛋白完全结合化合物)。将人BTK蛋白变性，离心沉淀。采用LC-MS/MS或HPLC分析上清液中原化合物的含量。在S8前孵育期间，无论BTK蛋白是否存在，B43都保持在上清中，这与BTK结合的可逆性一致(图3B)。相比之下，31a只在不含BTK蛋白的预孵育下从上清中恢复。用BTK预孵育后，31a大部分从上清中消失(图3A)，这与31a与BTK蛋白共价/不可逆结合的概念一致，因此用沉淀的BTK蛋白从上清中去除。数据还表明，BTK与31a之间的共价键形成基本在5分钟内完成[4]。

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在生化和细胞实验中，BGB-3111显示出纳米级的BTK抑制活性。在一些MCL和DLBCL细胞系中，BGB-3111抑制BCR聚集引发的BTK自磷酸化，阻断下游PLC-γ2信号传导，并有效抑制细胞增殖。与依鲁替尼相比，BGB-3111对一组激酶(包括ITK)显示出更有限的脱靶活性。虽然伊鲁替尼显著抑制利妥昔单抗诱导的NK细胞IFN-γ分泌和对套细胞淋巴瘤细胞的体外细胞毒性，但BGB-3111对利妥昔单抗诱导的ADCC的抑制作用至少弱于伊鲁替尼的10倍，这与其较弱的ITK抑制活性一致。[2]

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微粒体内部清除率:[4]
化合物(1µM)以蛋白浓度0.5 mg/mL孵育于肝微粒体中。人分别在反应开始后0、2、6、10、20、30 min，狗、大鼠、小鼠分别在反应开始后0、5、10、20、40、60 min分别以S50终止反应。采用LC-MS/MS对化合物进行分析。化合物剩余百分比的自然对数与孵育时间作了对比。内禀清除率(CLint)由下式计算，其中c蛋白为孵育系统中的微粒体蛋白浓度。CLint = -slope/Cprotein.[4]

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细胞色素P450的抑制作用[4]
采用8种CYP探针底物测定混合人肝微粒体的IC50值，并采用LC-MS/MS方法对样品进行分析，评价化合物对人CYP的抑制潜力。不同浓度的化合物与每个探针底物在混合的人肝微粒体中孵育。采用选择性抑制剂作为阳性对照来验证培养系统。监测探针代谢物的形成速率，计算IC50值。

BGB-3111 (31a, Zanubrutinib)的细胞毒性评价(HEK293和Ramos增殖试验):采用celltir - glo荧光细胞活力法测定化合物对HEK293和Ramos的生长抑制活性。96孔板每孔的细胞数量对每个细胞系进行优化，以确保在6天的处理期间呈对数增长(HEK293细胞1000个/孔，Ramos细胞6000个/孔)。细胞用10点稀释系列处理三次。暴露于该化合物6天后，加入体积等于每孔中细胞培养基体积的CellTiter-Glo试剂。混合物在轨道振动器上混合5分钟，使细胞裂解，然后在室温下孵育10分钟，使发光信号形成和稳定，这与ATP的数量相对应，从而与代谢活跃细胞的数量相对应。用PHERAstar FS阅读器测量发光信号。使用GraphPad Prism软件测定细胞活力的IC50值。[4]

Pharmacokinetics (PK) study in rats: [4]
Sprague-Dawley rats were housed in temperature (20-25°C) and humidity (40-70%) controlled facility with 12-hour light/dark cycle. The rats were sterilized water. The animals were fasted overnight prior to dosing until 4 hours post dosing. Water was not restricted during the study. Blood samples (0.15 mL) were collected via jugular vein cannula into dry heparinized (coated with 0.25% heparin in saline) tubes. For the IV administered groups, the sampling time points were pre-dose, 5, 15, 30, 45 min, 1, 2, 3, 4 and 6 h post-dose. For the oral administered groups, the sampling time points were pre-dose 15, 30 min, 1, 2, 3, 4, 6, 8, 12, and 24 h post-dose. Plasma samples were separated via centrifugation at 5500 rpm for 10 min, and stored in -30°C freezer until analysis. The plasma samples were analysis using LC-MS/MS with low limit of quantification (LLOQ) at 1 ng/mL. All PK parameters were calculated with non-compartment analysis using Phoenix WinNonlin 6.3.

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Plasma protein binding: [4]
The plasma protein binding of compounds was measured using equilibrium dialysis with HTDialysis equilibrium dialysis chamber apparatus. In brief, an aliquot of compounds spiked plasma was added to the donor side of each designated well. An equal volume of phosphate buffer (0.002% Tween 80) was added to the receiver side. The plate was covered with adhesive sealing film to prevent evaporation and placed in a water bath at 37ºC for 5-8 h with a shaking speed at 80 rpm to reach protein binding equilibrium. Samples were taken from both sides, and analyzed using LC-MS/MS. The value of unbound fraction (fu) was determined by dividing the compounds concentration on the buffer side of the equilibrium dialysis chamber by the compounds concentration on the plasma side.[4]

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In mouse BTK occupancy assays, treatment with BGB-3111 resulted in a dose-dependent BTK occupancy and showed about 3-fold more potency than ibrutinib in target organs, including PBMC and spleen. BGB-3111 induced dose-dependent anti-tumor effects against REC-1 MCL xenografts engrafted either subcutaneously or systemically via tail vein injection in mice. In the subcutaneous xenografts, BGB-3111 at 2.5 mg/kg BID showed similar activity as ibrutinib at 50 mg/kg QD, its clinical relevant dose. In the systemic model, the median survival of BGB-3111 25 mg/kg BID group was significantly longer than those of both ibrutinib 50 mg/kg QD and BID groups. In an ABC-subtype DLBCL (TMD-8) subcutaneous xenograft model, BGB-3111 also demonstrated better anti-tumor activity than ibrutinib. Preliminary 14-day toxicity study in rats showed that BGB-3111 was very well tolerated and maximal tolerate dose (MTD) was not reached when it was dosed up to 250mg/kg/day.[2]

Absorption, Distribution and Excretion
Following oral administration of zanubrutinib 160 mg twice daily and 320 mg once daily, the mean (%CV) zanubrutinib steady-state concentrations were 2,295 (37%) ng·h/mL and 2,180 (41%) ng·h/mL, respectively. The mean Cmax (%CV) was 314 (46%) ng/mL following 160 mg twice daily and 543 (51%) ng/mL following 320 mg once daily. The Cmax and AUC of zanubrutinib increase in a dose-proportional manner and there is minimal systemic accumulation after repeated dosing. The median Tmax is 2 hours.
Following oral administration of 320 mg radiolabelled zanubrutinib, approximately 87% of the dose was excreted in the feces and about 8% of the dose was recovered in the urine, where less than 1% of the recovered drug comprised of unchanged parent drug.
The geometric mean (%CV) apparent steady-state Vd is 881 (95%) L. The blood-to­ plasma ratio is about 0.7 to 0.8.
The mean (%CV) apparent oral clearance (CL/F) of zanubrutinib is 182 (37%) L/h.

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Metabolism / Metabolites
Zanubrutinib is predominantly metabolized by CYP3A4. Its metabolites have not been characterized.

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Biological Half-Life
Following administration of a single oral dose of 160 mg or 320 mg of zanubrutinib, the mean half-life is approximately 2 to 4 hours.

Hepatotoxicity
In the prelicensure clinical trials of zanubrutinib in patients with mantle cell lymphoma, liver test abnormalities were frequent although usually mild. ALT elevations arose in 28% and bilirubin levels in 24% of subjects, but were above 5 times the upper limit of normal (ULN) in less than 1%. In these trials that enrolled over 600 patients, there were no reports of clinically apparent liver injury, early discontinuations because of liver injury or liver related deaths. Nonetheless, other Bruton’s kinase inhibitors (ibrutinib, acalabrutinib) have been associated with rare cases of acute liver injury including acute liver failure. With those agents, the latency to onset of liver injury varied from several weeks to 9 months. The injury was typically hepatocellular and immunologic features were uncommon. While the pattern of injury was hepatocellular, the course was atypical of an acute hepatitis-like injury and more similar to acute hepatic necrosis with early onset of hepatic failure. Other Bruton’s kinase inhibitors have also been linked to several instances of reactivation of hepatitis B that can be severe and has been linked to fatal outcomes.
Likelihood score: E* (unproven but suspected cause of clinically apparent liver injury including reactivation of hepatitis B in susceptible patients).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of zanubrutinib during breastfeeding. Because zanubrutinib is 94% bound to plasma proteins, the amount in milk is likely to be low. The manufacturer recommends that breastfeeding be discontinued during zanubrutinib therapy and for at least 2 weeks 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
The plasma protein binding of zanubrutinib is approximately 94%.

[1]. Cancer Cell. 2015 Aug 10;28(2):225-39.

[2]. Cancer Res (2015) 75 (15_Supplement): 2597.

[3]. J Hematol Oncol . 2016 Sep 2;9(1):80.

[4]. J Med Chem . 2019 Sep 12;62(17):7923-7940.

Pharmacodynamics
Zanubrutinib is an immunomodulating agent that decreases the survival of malignant B cells. It inhibits BTK by binding to its active site. It works to inhibit the proliferation and survival of malignant B cells to reduce the tumour size in mantle cell lymphoma.

C27H29N5O3

471.56

471.227

C, 68.77; H, 6.20; N, 14.85; O, 10.18

1691249-45-2

(±)-Zanubrutinib;1633350-06-7;(R)-Zanubrutinib;1691249-44-1;Zanubrutinib-d5

135565884

White to off-white solid powder

1.3±0.1 g/cm3

713.4±60.0 °C at 760 mmHg

385.2±32.9 °C

0.0±2.3 mmHg at 25°C

1.680

3.64

103

2

5

6

35

756

1

O=C(C=C)N1CCC(CC1)[C@]1([H])CCNC2=C(C(N)=O)C(C3C=CC(=CC=3)OC3C=CC=CC=3)=NN12

RNOAOAWBMHREKO-QFIPXVFZSA-N

InChI=1S/C27H29N5O3/c1-2-23(33)31-16-13-18(14-17-31)22-12-15-29-27-24(26(28)34)25(30-32(22)27)19-8-10-21(11-9-19)35-20-6-4-3-5-7-20/h2-11,18,22,29H,1,12-17H2,(H2,28,34)/t22-/m0/s1

(7S)-2-(4-phenoxyphenyl)-7-(1-prop-2-enoylpiperidin-4-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide

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: ≥ 2.08 mg/mL (4.41 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 20.8 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 2 中的溶解度: ≥ 2.08 mg/mL (4.41 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 20.8 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: ≥ 2.08 mg/mL (4.41 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 20.8 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;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。