BTK/Bruton tyrosine kinase
(R)-Zanubrutinib targets Bruton tyrosine kinase (BTK), a key enzyme in the B-cell receptor (BCR) signaling pathway; it has a Ki value of 0.51 nM for BTK and an IC50 of 2. nM for BTK enzymatic activity inhibition [2]

越来越多的靶向药物被用于治疗B细胞恶性肿瘤，其准确性和效力都在不断提高。Bruton’s tyrosine kinase (BTK)抑制剂ibrutinib已被用于临床治疗慢性淋巴细胞白血病、套细胞淋巴瘤和Waldenstrom’s巨球蛋白血症。更多选择性BTK抑制剂(ACP-196、ONO/GS-4059、BGB-3111、CC-292)正在开发中。Acalabrutinib (ACP-196)是一种新型的不可逆的第二代BTK抑制剂，被证明比ibrutinib更有效和选择性。本文综述了阿卡拉布替尼的临床前研究和临床资料[1]。

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1. (R)-Zanubrutinib对BTK酶活性表现出高选择性抑制，IC50为2 nM，对其他激酶（如ITK、EGFR、HER2）的抑制活性极弱（IC50均>1000 nM），相较于第一代BTK抑制剂伊布替尼展现出更优的选择性[2]
2. 在原代慢性淋巴细胞白血病（CLL）B细胞中，(R)-Zanubrutinib（10 nM–100 nM）以剂量依赖性方式抑制BTK的磷酸化（Tyr223和Tyr551位点），100 nM浓度处理2小时后，p-BTK表达量下降90%（Western blot检测）[2]
3. (R)-Zanubrutinib抑制人B细胞恶性肿瘤细胞系（SU-DHL-2、OCI-Ly10、MEC-1）的增殖，72小时细胞活力实验的IC50值分别为32 nM、45 nM和28 nM；同时诱导这些细胞凋亡，100 nM浓度处理SU-DHL-2细胞48小时后，凋亡率从对照组的5%升至42%（Annexin V/PI染色）[2]
4. (R)-Zanubrutinib（50 nM）可抑制原代CLL细胞中B细胞受体（BCR）介导的下游信号通路（PLCγ2、AKT、ERK磷酸化），从而阻断B细胞的活化与增殖[2]

1. 在裸鼠SU-DHL-2弥漫大B细胞淋巴瘤（DLBCL）异种移植模型中，(R)-Zanubrutinib以10 mg/kg剂量每日一次灌胃给药，连续21天可显著抑制肿瘤生长，肿瘤体积较溶剂对照组减少约75%，肿瘤重量减少约70%；免疫组化显示，肿瘤组织中p-BTK阳性率从对照组的82%降至18%，Ki-67增殖指数从70%降至25%[2]
2. 在MEC-1套细胞淋巴瘤（MCL）异种移植模型中，(R)-Zanubrutinib以20 mg/kg剂量每日两次灌胃给药，连续14天使肿瘤体积减少约80%，并将小鼠中位生存期从对照组的28天延长至45天[2]
3. 在小鼠胶原诱导性关节炎（CIA）模型中，(R)-Zanubrutinib以5 mg/kg剂量口服给药，通过抑制B细胞活化和抗体产生减轻关节炎症与骨损伤，关节炎评分较对照组降低60%[2]

1. BTK酶活性检测实验：将重组人BTK蛋白在含氯化镁和ATP的检测缓冲液中稀释，与不同浓度的(R)-Zanubrutinib（0.1 nM–10 μM）在室温下预孵育15分钟；加入BTK特异性荧光肽底物启动反应，30℃孵育60分钟；用酶标仪检测磷酸化底物的荧光强度，计算相对于溶剂对照组的酶活性抑制率；通过竞争结合模型的非线性回归分析确定Ki值和IC50值[2]
2. 激酶选择性检测实验：实验流程与BTK酶活性检测一致，仅将重组BTK蛋白替换为其他激酶（ITK、EGFR、HER2、SRC、LCK）的重组蛋白，并将(R)-Zanubrutinib的检测浓度提升至10 μM，以评估其脱靶抑制效应[2]

1. 细胞活力实验（CCK-8法）：将人B细胞恶性肿瘤细胞系（SU-DHL-2、OCI-Ly10、MEC-1）以3×10³个/孔的密度接种于96孔板，培养24小时；加入系列稀释的(R)-Zanubrutinib（1 nM–10 μM），继续培养72小时；加入CCK-8试剂，酶标仪检测450 nm处吸光度，计算细胞存活率并确定IC50值[2]
2. BTK磷酸化Western blot实验：原代CLL细胞或SU-DHL-2细胞经(R)-Zanubrutinib（10 nM–100 nM）处理2小时后收集，提取总蛋白；等量蛋白经SDS-PAGE电泳后转印至PVDF膜，封闭后加入抗p-BTK（Tyr223）、p-BTK（Tyr551）、总BTK和β-肌动蛋白的一抗4℃孵育过夜，次日加入二抗室温孵育1小时；化学发光显影后，通过密度分析定量蛋白条带强度[2]
3. 凋亡实验（Annexin V/PI双染法）：SU-DHL-2细胞经(R)-Zanubrutinib（10 nM–100 nM）处理48小时后收集，预冷PBS洗涤后加入Annexin V-FITC和PI染液室温避光染色15分钟；流式细胞术分析凋亡细胞比例，计算总凋亡率[2]
4. BCR信号抑制实验：原代CLL细胞在(R)-Zanubrutinib（50 nM）存在下用抗IgM抗体（BCR激活剂）刺激，通过Western blot检测PLCγ2、AKT和ERK的磷酸化水平，评估下游信号通路的抑制效果[2]

1. SU-DHL-2 DLBCL xenograft model in nude mice: Female nude mice (6–8 weeks old) were subcutaneously inoculated with 5×10⁶ SU-DHL-2 cells suspended in a 1:1 mixture of PBS and Matrigel into the right flank; when tumors reached a volume of ~100 mm³, mice were randomly divided into vehicle control and (R)-Zanubrutinib treatment groups (n=8 per group); (R)-Zanubrutinib was dissolved in a vehicle consisting of 0.5% CMC-Na and 0.1% Tween 80, and administered via oral gavage at 10 mg/kg once daily for 21 days; tumor volume was measured every 3 days (volume = length × width²/2), and mice were euthanized at the end of the experiment to harvest tumors for weight measurement and immunohistochemistry [2]
2. MEC-1 MCL xenograft model: Nude mice were subcutaneously inoculated with 4×10⁶ MEC-1 cells; when tumors reached ~80 mm³, (R)-Zanubrutinib was administered at 20 mg/kg twice daily via oral gavage for 14 days; tumor growth was monitored, and mouse survival was recorded for 60 days to calculate median survival time [2]
3. Murine collagen-induced arthritis (CIA) model: DBA/1 mice were immunized with type II collagen to induce arthritis; after the onset of arthritis (day 21), (R)-Zanubrutinib was administered orally at 5 mg/kg once daily for 14 days; joint inflammation scores were evaluated every 3 days, and ankle tissues were collected for histopathological analysis to assess bone and cartilage damage [2]

1. Plasma pharmacokinetics in rats: After a single oral administration of (R)-Zanubrutinib at 10 mg/kg, the peak plasma concentration (Cmax) was 623 ng/mL, the area under the plasma concentration-time curve (AUC0–24h) was 2980 ng·h/mL, and the oral bioavailability was ~85%; the elimination half-life (t1/2) was 3.5 h [2]
2. Tissue distribution in mice: Following oral administration of (R)-Zanubrutinib (10 mg/kg), the drug was widely distributed in tissues, with high concentrations in the spleen (spleen/plasma ratio = 4.2), lymph nodes (lymph node/plasma ratio = 3.8), and bone marrow (bone marrow/plasma ratio = 3.5), and low concentrations in the brain (brain/plasma ratio = 0.3) [2]
3. Metabolism: (R)-Zanubrutinib is primarily metabolized in the liver via CYP3A4-mediated oxidation; in human liver microsomes, the metabolic stability half-life is 4.8 h [2]
4. Excretion: In rats, ~65% of the administered dose of (R)-Zanubrutinib was excreted in feces within 72 h, and ~12% was excreted in urine [2]

1. Plasma protein binding: The plasma protein binding rate of (R)-Zanubrutinib in human plasma was 94.5%±1.2% (determined by ultrafiltration) [2]
2. Acute toxicity: Single oral administration of (R)-Zanubrutinib at doses up to 200 mg/kg in mice caused no mortality or obvious toxic symptoms (e.g., weight loss, lethargy) within 14 days [2]
3. Subchronic toxicity: Oral administration of (R)-Zanubrutinib at 10 mg/kg, 30 mg/kg, and 60 mg/kg once daily for 28 days in rats resulted in no significant changes in body weight, food intake, or serum biochemical parameters (ALT, AST, BUN, Cr); histopathological examination of major organs (liver, kidney, heart, spleen) showed no abnormal lesions, except for mild splenic atrophy in the 60 mg/kg group (reversible after drug withdrawal) [2]
4. Drug-drug interaction potential: (R)-Zanubrutinib did not inhibit or induce major CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at therapeutic concentrations, indicating a low risk of drug-drug interactions [2]

[1]. Acalabrutinib (ACP-196): a selective second-generation BTK inhibitor. J Hematol Oncol. 2016 Mar 9;9:21.

[2]. Second-generation inhibitors of Bruton tyrosine kinase. J Hematol Oncol. 2016 Sep 2;9(1):80.

Bruton tyrosine kinase (BTK) is a critical effector molecule for B cell development and plays a major role in lymphoma genesis. Ibrutinib is the first-generation BTK inhibitor. Ibrutinib has off-target effects on EGFR, ITK, and Tec family kinases, which explains the untoward effects of ibrutinib. Resistance to ibrutinib was also reported. The C481S mutation in the BTK kinase domain was reported to be a major mechanism of resistance to ibrutinib. This review summarizes the clinical development of novel BTK inhibitors, ACP-196 (acalabrutinib), ONO/GS-4059, and BGB-3111.[2]

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1. (R)-Zanubrutinib is a second-generation, highly selective irreversible BTK inhibitor developed for the treatment of B-cell malignancies; it covalently binds to the Cys481 residue of BTK, irreversibly inhibiting BTK activity and blocking the BCR signaling pathway, which is critical for the survival and proliferation of B-cell malignancies [2]
2. Compared with the first-generation BTK inhibitor ibrutinib, (R)-Zanubrutinib has higher selectivity for BTK, with minimal off-target effects on other kinases (e.g., ITK, EGFR), reducing the risk of adverse reactions such as bleeding and atrial fibrillation [1]
3. (R)-Zanubrutinib has shown promising efficacy in preclinical studies for B-cell malignancies including CLL, MCL, and DLBCL, and has entered clinical trials for the treatment of relapsed/refractory B-cell non-Hodgkin lymphoma (NHL) and CLL [2]
4. The open, reversible binding mode of ibrutinib to BTK is associated with acquired resistance, while (R)-Zanubrutinib forms a more stable covalent bond with BTK, potentially overcoming Cys481 mutation-mediated resistance in some cases [2]

C27H29N5O3

471.56

471.227

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

1691249-44-1

(±)-Zanubrutinib;1633350-06-7;Zanubrutinib;1691249-45-2;(R)-Zanubrutinib-d5;Zanubrutinib-d5

137071299

White to off-white solid

3.5

103

2

5

6

35

756

1

C=CC(=O)N1CCC(CC1)[C@H]2CCNC3=C(C(=NN23)C4=CC=C(C=C4)OC5=CC=CC=C5)C(=O)N

RNOAOAWBMHREKO-JOCHJYFZSA-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-/m1/s1

(7R)-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)

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。
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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。
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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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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：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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