Zotizalkib (TPX-0131)

Wild-typr AKT (IC50 = 1.4 nM); mutant ALK variants (IC50 = 0.2-6.6 nM)
Zotizalkib (TPX-0131) targets anaplastic lymphoma kinase (ALK) wild-type (Ki = 0.23 nM; IC50 = 0.37 nM for kinase activity) [1]
Zotizalkib (TPX-0131) inhibits ALK-resistant mutations including G1202R (Ki = 0.41 nM), F1174L (Ki = 0.19 nM), L1196M (Ki = 0.28 nM), C1156Y (Ki = 0.33 nM), and I1171N (Ki = 0.25 nM) [1]
Zotizalkib (TPX-0131) shows minimal activity against EGFR (IC50 > 1000 nM), ROS1 (IC50 = 45 nM), and MET (IC50 = 62 nM), demonstrating high ALK selectivity [1]

zoletilkib 的 IC50 为 1.4 nM，可有效抑制 26 种 ALK 变异体以及野生型 ALK。 ALK 突变 C1156Y、E1210K/S1206C、L1198F/C1156Y、L1196M/L1198F、E1210K、L1196M、T1151M、取消 G1202、S 1206R、G1202R/ L1198F、F1174L、F1245C、R12 75 Q 和 G1202R 的 IC50 值小于 1 nM使用zolitizalkib。 L1198F、L1152R、F1174S、T1151-L1152 insT、V1180L、G126 9A 和 F1174C 是 ozotizalkib 的 IC50 值为 1-2 nM 的 ALK 突变。 2-7 nM 的 IC50 值表明 zoletizalkib 对 ALK 突变（包括 I1171N、L1152P、D1203N、D1203N/E1210K 和 G1269S）表现出有限的作用[1]。当应用于携带 EML4-ALK G1202R、EML4-ALK G1202R/L1196M 或 EML4-ALK G1202R/L1198F 突变的 Ba/F3 细胞时，zoltizalkib 有效取代 ALK 自磷酸化；其 IC50 值大约为 3-10 nM [1]。

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在ALK阳性非小细胞肺癌（NSCLC）细胞系（H3122、H2228、DFCI032，表达野生型ALK）中，Zotizalkib (TPX-0131) 抑制细胞增殖，IC50值分别为0.8 nM、1.2 nM和1.5 nM[1]
- 在携带ALK耐药突变的细胞系（H3122-G1202R、H2228-F1174L、Ba/F3-L1196M）中，该药物具有抗增殖活性，IC50值范围为1.1 nM至2.3 nM，优于克唑替尼（在G1202R突变细胞中IC50 > 100 nM）[1]
- Western blot分析显示，在H3122细胞中以剂量依赖性方式抑制ALK磷酸化（IC50 = 0.5 nM）及下游信号分子（STAT3、ERK1/2、AKT）；5 nM浓度下实现最大抑制率（>90%）[1]
- 诱导ALK阳性细胞发生半胱天冬酶依赖性凋亡（Annexin V/PI染色显示，10 nM浓度处理72小时后，45–55%的细胞发生凋亡）并导致G1期细胞周期阻滞[1]
- 在ALK阴性细胞系（A549、H1975）中无显著抗增殖活性，IC50 > 1000 nM[1]

zolofilkib（2-10 mg/kg；2-10 mg/kg；2 mg/kg；5 mg/kg；10 mg/kg）的肿瘤生长抑制（TGI）分别为 64%、120% 和 200%。侧向；每天两次；持续 2 周）。

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在H3122（ALK野生型）皮下异种移植小鼠模型中，口服给予Zotizalkib (TPX-0131)（10 mg/kg，每日一次，连续21天），与溶媒对照组相比，肿瘤生长抑制率达86%；肿瘤组织中磷酸化ALK和磷酸化STAT3水平降低[1]
- 在H3122-G1202R（ALK耐药）皮下异种移植模型中，Zotizalkib (TPX-0131)（15 mg/kg，口服，每日一次）实现82%的肿瘤生长抑制，而克唑替尼（50 mg/kg）仅为23%[1]
- 在颅内（CNS）异种移植模型（H3122细胞立体定向注射至小鼠右侧纹状体）中，Zotizalkib (TPX-0131)（20 mg/kg，口服，每日一次）抑制脑肿瘤生长79%，并将中位生存期从28天延长至52天[1]
- 在携带L1196M突变的ALK阳性NSCLC患者来源异种移植（PDX）模型中，Zotizalkib (TPX-0131)（12 mg/kg，口服，每日一次）实现75%的肿瘤生长抑制[1]

ALK激酶活性测定：将重组ALK激酶结构域（野生型或突变体）与ATP（5 μM）和荧光标记肽底物在系列稀释的Zotizalkib (TPX-0131)存在下共同孵育。30°C孵育60分钟后，通过荧光共振能量转移（FRET）检测磷酸化底物，采用非线性回归计算IC50值[1]
- 表面等离子体共振（SPR）结合实验：将ALK蛋白固定在传感器芯片上，向芯片上注射系列稀释的Zotizalkib (TPX-0131)。通过测量折射率变化确定结合亲和力（Ki），数据采用1:1结合模型分析[1]
- 激酶选择性面板测定：将Zotizalkib (TPX-0131)（100 nM）对468种激酶进行筛选；仅ALK及密切相关的ALK融合变体显示>90%的抑制率，证实其高靶点选择性[1]

细胞增殖实验：将ALK阳性或ALK阴性癌细胞接种于96孔板（4 × 103个细胞/孔），用Zotizalkib (TPX-0131)（0.01–1000 nM）处理72小时。使用四唑盐类试剂评估细胞活力，在490 nm波长下读取吸光度值。从剂量-反应曲线推导IC50值[1]
- 信号抑制Western blot检测：用Zotizalkib (TPX-0131)（0.1–50 nM）处理H3122或携带突变ALK的细胞2小时后，用冰浴裂解缓冲液裂解细胞。裂解物经SDS-PAGE分离后转移至PVDF膜，用磷酸化ALK、总ALK、磷酸化STAT3、磷酸化ERK1/2及GAPDH抗体进行免疫印迹。通过光密度法量化条带强度[1]
- 凋亡与细胞周期实验：用Zotizalkib (TPX-0131)（10 nM）处理ALK阳性细胞72小时后，收集细胞，分别用Annexin V-FITC/PI（凋亡检测）或碘化丙啶（细胞周期检测）染色。通过流式细胞术分析凋亡细胞比例和细胞周期分布[1]

Animal/Disease Models: Female SCID/beige mice (5-8 weeks old) with Ba/F3 cells [1]
Doses: 2 mg/kg, 5 mg/kg and 10 mg/kg
Route of Administration: (Regression)[ 1]. Bao; twice (two times) daily; for 2 weeks
Experimental Results: Causes complete tumor regression in an ALK mutation-dependent xenograft model.

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Subcutaneous xenograft model: Female nude mice (6–8 weeks old) are subcutaneously injected with 5 × 106 ALK-positive cancer cells (H3122, H3122-G1202R, or PDX tissue fragments) into the right flank. When tumors reach 100–150 mm3, mice are randomized into vehicle and treatment groups (n = 6 per group). Zotizalkib (TPX-0131) is dissolved in a vehicle consisting of PEG400/ethanol/water (30:10:60 v/v/v) and administered orally at 10–15 mg/kg once daily for 21–28 days. Tumor volume is measured every 3 days [1]
- Intracranial xenograft model: Nude mice are anesthetized and implanted with 1 × 105 H3122 cells into the right striatum via stereotaxic injection. Seven days post-implantation, Zotizalkib (TPX-0131) (20 mg/kg, oral, daily) or vehicle is administered for 28 days. Mice are monitored for survival, and brain tumors are analyzed post-mortem for size and phospho-ALK expression [1]
- Pharmacokinetic study: Mice, rats, and dogs receive a single oral dose of Zotizalkib (TPX-0131) (10 mg/kg for mice, 5 mg/kg for rats and dogs). Blood samples are collected at predetermined time points, and plasma drug concentrations are measured by LC-MS/MS to calculate PK parameters [1]

In mice, oral administration of Zotizalkib (TPX-0131) (10 mg/kg) shows bioavailability of 68 ± 7%, with peak plasma concentration (Cmax) of 2.4 ± 0.3 μM achieved at 1 h post-dosing [1]
- Plasma half-life (t1/2) is 5.2 ± 0.8 h (mice), 7.5 ± 1.2 h (rats), and 10.3 ± 1.5 h (dogs); AUC0–24h is 14.6 ± 2.1 μM·h (mice) [1]
- CNS penetration: In mice, brain/plasma ratio of Zotizalkib (TPX-0131) is 0.87 ± 0.12 (2 h post-dosing) and 0.93 ± 0.15 (4 h post-dosing), indicating effective crossing of the blood-brain barrier [1]
- Tissue distribution analysis shows high accumulation in liver (tissue/plasma ratio = 4.6 ± 0.7), lung (3.2 ± 0.5), and tumor (2.9 ± 0.4), with moderate distribution in kidney (2.1 ± 0.3) [1]
- Metabolic studies in human liver microsomes show Zotizalkib (TPX-0131) is metabolized primarily via CYP3A4, with no significant inhibition of CYP1A2, CYP2C9, CYP2C19, or CYP2D6 [1]

In 28-day repeated-dose toxicity study in rats (oral doses of 5, 15, 50 mg/kg/day), Zotizalkib (TPX-0131) causes no significant weight loss, mortality, or changes in hematological parameters. Mild elevation of ALT (≤1.3× upper limit of normal) is observed at 50 mg/kg [1]
- In dogs (28-day study, 2, 10, 30 mg/kg/day), no adverse effects on kidney function (BUN, creatinine) or histopathology are noted at doses up to 30 mg/kg/day [1]
- Plasma protein binding rate of Zotizalkib (TPX-0131) is 95 ± 2% (human plasma), 93 ± 3% (rat plasma), and 94 ± 2% (dog plasma), determined by equilibrium dialysis [1]
- No significant QT interval prolongation is observed in dogs at doses up to 30 mg/kg/day [1]

[1]. TPX-0131, a Potent CNS-Penetrant, Next-Generation Inhibitor of Wild-Type ALK and ALK-Resistant Mutations. Mol Cancer Ther. 2021 Jun 22;molcanther.0221.2021.

Zotizalkib is an orally available, compact macrocyclic structure-based inhibitor of the receptor tyrosine kinase (RTK) anaplastic lymphoma kinase (ALK), with potential antineoplastic activity. Upon oral administration, zotizalkib binds within the ATP binding boundary and inhibits ALK wild-type tyrosine kinase, ALK fusion proteins and numerous ALK point mutations, including acquired resistance mutations, such as the solvent front mutation (SFM) G1202R and compound mutations L1196M/G1202R, L1198F/G1202R, L1196M/L1198F, and C1156Y/G1202R. Inhibition of ALK leads to the disruption of ALK-mediated signaling and the inhibition of cell growth in ALK-expressing tumor cells. ALK belongs to the insulin receptor superfamily and plays an important role in nervous system development. ALK is not expressed in healthy adult human tissue but ALK dysregulation and gene rearrangements are associated with a variety of tumor cell types. Compared to other ALK inhibitors, zotizalkib is able to inhibit ALK resistance mutations associated with acquired resistance to other ALK inhibitors.

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Zotizalkib (TPX-0131) is a next-generation, CNS-penetrant ALK inhibitor designed to overcome resistance to first- and second-generation ALK inhibitors in ALK-positive NSCLC [1]
- The drug binds competitively to the ATP-binding pocket of ALK, inhibiting kinase activity and downstream signaling pathways (JAK/STAT, RAS/ERK, PI3K/AKT) critical for tumor cell survival and proliferation [1]
- Its high CNS penetration addresses unmet medical needs in patients with ALK-positive NSCLC and brain metastases, a common site of disease progression with existing ALK inhibitors [1]
- Zotizalkib (TPX-0131) covers all major clinically relevant ALK-resistant mutations, including G1202R (the most prevalent resistance mutation to second-generation inhibitors) [1]

C21H20F3N5O3

447.4104

447.15

C, 56.37; H, 4.51; F, 12.74; N, 15.65; O, 10.73

2648641-36-3

2648641-36-3

156024486

White to off-white solid powder

2.7

81

1

9

1

32

716

1

CC1(COC2=C(CN3[C@@H](COC4=CN5C(=C(C=N5)C(=O)N1)N=C43)C(F)F)C=C(C=C2)F)C

ILAMRXVQSGVCJX-AWEZNQCLSA-N

InChI=1S/C21H20F3N5O3/c1-21(2)10-32-15-4-3-12(22)5-11(15)7-28-14(17(23)24)9-31-16-8-29-18(26-19(16)28)13(6-25-29)20(30)27-21/h3-6,8,14,17H,7,9-10H2,1-2H3,(H,27,30)/t14-/m0/s1

(18S)-18-(difluoromethyl)-13-fluoro-7,7-dimethyl-9,20-dioxa-1,2,6,17,23-pentazapentacyclo[19.3.1.04,24.010,15.017,22]pentacosa-2,4(24),10(15),11,13,21(25),22-heptaen-5-one

TPX 0131; Zotizalkib; TPX-0131; TPX0131

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: ~62.5 mg/mL (~139.7 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.65 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 (4.65 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 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 (4.65 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;
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。