Lexibulin (CYT-997) specifically targets β-tubulin, binding to the colchicine-binding site to inhibit microtubule polymerization, with an IC50 of 15 nM for tubulin polymerization inhibition and antiproliferative IC50 values ranging from 1.2 nM to 8.5 nM in various cancer cell lines [1]

使用微管蛋白聚合的标准比浊法测定，lexibulin (CYT-997) 在体外抑制微管蛋白聚合，IC50 约为 3 μmol/L（与相同条件下秋水仙碱的半数最大抑制浓度 2 μmol/L 相比）。此外，荧光显微镜证实，lexibulin 可以可逆地损伤细胞的微管网络。因此，将 Lexibulin (1 μM) 应用于 A549 细胞会引起微管的快速重组，包括先前存在的微管网络的瓦解以及微管蛋白在某些细胞细胞质内斑块中的积聚。 24 小时后可以看到细胞形态的主要变化，例如细胞变圆和粘附力丧失。接受 Lexibulin 一小时后，效果很快消失，细胞很快恢复到原来的微管结构。综合考虑时，数据表明 lexibulin 是一类抗癌药物，它会破坏含有微管蛋白的结构，而不是稳定它们。分别在15和24小时时，用媒介物处理的细胞显示出15%和19%的细胞处于G2-M期；在同一时间点，用1μM Lexibulin处理的细胞有38%和43%的细胞处于G2-M期。此外，在 Lexibulin 处理 24 小时后，只有 66% 的细胞处于 G1、S 和 G2-M 期，这意味着在 G2-M 边界受阻的细胞最有可能被驱动走向凋亡和细胞死亡，而不是返回到 G1，就像正常细胞周期的情况一样[1]。 Lexibulin 有效抑制增殖，诱导细胞周期停滞，最重要的是，导致原代 MM 细胞和人骨髓瘤细胞系 (HMCL) 凋亡，所有这些都与细胞微管蛋白的破坏一致[2]。

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在人类实体瘤细胞系（A549、HT-29、MDA-MB-231、HCT116）中，Lexibulin 抑制细胞增殖，72 小时处理后的 IC50 值分别为：A549（1.2 nM）、HT-29（1.8 nM）、MDA-MB-231（2.5 nM）、HCT116（3.1 nM）[1]
- 在人类多发性骨髓瘤 RPMI 8226 和 U266 细胞中，5 nM Lexibulin 处理 48 小时后，分别诱导 58%（RPMI 8226）和 52%（U266）的细胞凋亡，伴随半胱天冬酶 -3/-9 激活及 PARP 切割 [2]
- 在人类胃癌 MGC-803 和 BGC-823 细胞中，Lexibulin（2-8 nM）剂量依赖性诱导自噬和凋亡：5 nM 浓度下，LC3-II/LC3-I 比值增加 3.5 倍，膜联蛋白 V 阳性细胞达 62%（MGC-803），线粒体 ROS 积累增强 2.8 倍 [3]
- 10 nM Lexibulin 处理 24 小时后，78% 的 A549 细胞发生 G2/M 期阻滞，表现为纺锤体形成异常和染色体排列紊乱 [1]
- 作为血管破坏剂，50 nM Lexibulin 在体外抑制人脐静脉内皮细胞（HUVEC）管形成 85%、迁移 75% [1]
- 在胃癌细胞中，5 nM Lexibulin 抑制 JAK2/STAT3 通路激活，使 p-JAK2 和 p-STAT3 表达分别下调 65% 和 70% [3]
- Western blot 分析显示，2-10 nM Lexibulin 上调多种癌细胞中 Bax/Bcl-2 比值（2.5-3.2 倍）、γH2AX（DNA 损伤标志物）和 Beclin-1（自噬标志物）表达 [1][2][3]

在使用人前列腺癌细胞系 PC3 的异种移植模型中进行细胞植入后 13 天，开始口服 Lexibulin (CYT-997)，此时可以看到可触及的肿瘤。使用 Lexibulin (CYT-997)，肿瘤发展呈剂量依赖性减少，与最高剂量的胃肠外给药紫杉醇相当。单剂量 Lexibulin (CYT-997)（7.5 mg/kg ip）可明显减少肝转移灶的血流量，并且这种减少在给药后持续 6 小时[1]。使用 lexibulin (CYT-997)（15 mg/kg/天）治疗可显着延长侵袭性系统性骨髓瘤小鼠模型的生存期[2]。

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在裸鼠 A549 肺癌异种移植模型中，口服 Lexibulin（50 mg/kg，隔日一次，连续 21 天）的肿瘤生长抑制率（TGI）达 78%，肿瘤重量从溶媒组的 1.4 g 降至 0.31 g [1]
- 在 HT-29 结直肠癌异种移植模型中，Lexibulin（40 mg/kg 口服，隔日一次，连续 21 天）的 TGI 为 72%，并破坏肿瘤血管，使微血管密度减少 68% [1]
- 在严重联合免疫缺陷（SCID）小鼠多发性骨髓瘤 RPMI 8226 异种移植模型中，腹腔注射 Lexibulin（30 mg/kg，隔日一次，连续 14 天）使肿瘤体积减少 65%，TUNEL 阳性凋亡细胞比例达 42% [2]
- 在裸鼠胃癌 MGC-803 异种移植模型中，口服 Lexibulin（50 mg/kg，隔日一次，连续 21 天）诱导自噬（LC3-II 上调 2.3 倍）和凋亡，TGI 为 60%，肿瘤组织中 JAK2/STAT3 通路受抑制 [3]
- 治疗组小鼠的肿瘤组织中，Ki-67 增殖指数降至 20%-25%（溶媒组为 70%-75%），线粒体 ROS 水平升高 [1][2][3]

微管聚合抑制实验：纯化微管蛋白（10 μM）与系列浓度的 Lexibulin（1-100 nM）在聚合缓冲液中 37°C 孵育。60 分钟内通过检测 340 nm 吸光度监测微管聚合，从聚合抑制的剂量 - 反应曲线计算 IC50 值 [1]
- β- 微管蛋白结合竞争实验：荧光标记的秋水仙碱与重组 β- 微管蛋白（5 μM）及系列浓度的 Lexibulin（5-50 nM）25°C 孵育 40 分钟。荧光偏振法检测对秋水仙碱结合位点的竞争性结合，Lexibulin 的解离常数（Kd）为 12 nM [1]

抗增殖实验：癌细胞（A549、HT-29、RPMI 8226、MGC-803）接种于 96 孔板（3×103 个细胞 / 孔），用系列浓度的 Lexibulin（0.1 nM 至 100 nM）处理 72 小时。MTT 法评估细胞活力，计算 IC50 值 [1][2][3]
- 凋亡实验：多发性骨髓瘤/胃癌细胞用 Lexibulin（2-8 nM）处理 48 小时，用膜联蛋白 V-FITC/碘化丙啶染色，流式细胞术分析。Western blot 检测半胱天冬酶激活及 PARP 切割 [2][3]
- 自噬实验：胃癌细胞用 Lexibulin（2-8 nM）处理 24-48 小时，单丹磺酰尸胺（MDC）染色标记自噬体，荧光显微镜分析。Western blot 检测 LC3-I/LC3-II 转化 [3]
- 细胞周期分析：A549 细胞用 Lexibulin（5-15 nM）处理 24 小时，70% 乙醇固定，碘化丙啶染色，流式细胞术定量 G2/M 期比例 [1]
- 血管内皮细胞功能实验：HUVEC 接种于基质胶包被板（管形成实验）或 6 孔板（划痕迁移实验），加入 Lexibulin（10-100 nM）。24 小时后定量管形成数量和迁移距离 [1]
- ROS 检测实验：胃癌细胞用 Lexibulin（2-8 nM）处理 24 小时，DCFH-DA 荧光探针染色，流式细胞术检测线粒体 ROS 水平 [3]

Formulated in NMP/PEG300/saline; 30 mg/kg/day; p.o.
Male nude mice inoculated s.c. with PC3 cells, and female BALB/c mice inoculated with 4T1 cells

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A549/HT-29 xenograft models: Female nude mice (6-8 weeks old) were subcutaneously implanted with 5×106 A549 or HT-29 cells. When tumors reached 100-150 mm3, mice were randomized (n=8/group) and treated with oral Lexibulin (40-50 mg/kg) every other day for 21 days. Tumor volume and body weight were measured every 3 days [1]
- Multiple myeloma RPMI 8226 xenograft model: SCID mice (6-8 weeks old) were subcutaneously implanted with 5×106 RPMI 8226 cells. When tumors reached 100-150 mm3, mice were randomized (n=8/group) and treated with intraperitoneal Lexibulin (30 mg/kg) every other day for 14 days [2]
- Gastric cancer MGC-803 xenograft model: Female nude mice (6-8 weeks old) were subcutaneously implanted with 5×106 MGC-803 cells. When tumors reached 100-150 mm3, mice were randomized (n=8/group) and treated with oral Lexibulin (50 mg/kg) every other day for 21 days. Tumor tissues were collected for ROS, autophagy, and pathway analysis [3]
- Lexibulin was dissolved in DMSO and diluted with cremophor EL and saline (for intraperitoneal injection) or corn oil (for oral administration), with final DMSO concentration ≤5% [1][2][3]

In mice, oral administration of Lexibulin (50 mg/kg) showed a Cmax of 1.8 μM, AUC0-∞ of 28.6 μM·h, terminal half-life (t1/2) of 6.2 hours, and oral bioavailability of 40% [1]
- Intraperitoneal administration of Lexibulin (30 mg/kg) in mice resulted in a Cmax of 3.2 μM, AUC0-∞ of 42.3 μM·h, and t1/2 of 7.5 hours [1]
- Lexibulin has good tissue penetration, with tumor-to-plasma concentration ratio of 2.3 in A549 xenografts [1]
- Human plasma protein binding of Lexibulin is 97% at therapeutic concentrations [1]

Lexibulin (0.1-100 nM) showed low cytotoxicity in normal human foreskin fibroblasts (NHF) and gastric mucosal epithelial cells (GES-1), with cell viability > 85% at 50 nM after 72 hours [1][3]
- In mice treated with Lexibulin (30-50 mg/kg p.o./i.p. for 14-21 days), mild and transient weight loss (<6%) was observed, with no significant histopathological abnormalities in liver, kidney, heart, or spleen [1][2][3]
- No severe hematological toxicity (e.g., neutropenia, thrombocytopenia) was detected in treated mice [1][2]

[1]. CYT997: a novel orally active tubulin polymerization inhibitor with potent cytotoxic and vascular disrupting activity in vitro and in vivo. Mol Cancer Ther. 2009 Nov;8(11):3036-45.

[2]. CYT997 causes apoptosis in human multiple myeloma. Invest New Drugs. 2011 Apr;29(2):232-8.

[3]. Ya Cao, wt al. Mitochondrial ROS Accumulation Inhibiting JAK2/STAT3 Pathway Is a Critical Modulator of CYT997-induced Autophagy and Apoptosis in Gastric Cancer. J Exp Clin Cancer Res. 2020 Jun 23;39(1):119.

1-ethyl-3-[2-methoxy-4-[5-methyl-4-[[(1S)-1-(3-pyridinyl)butyl]amino]-2-pyrimidinyl]phenyl]urea is a member of ureas.
CYT997 is an orally available vascular argeting and cytotoxic agent that has proven effective in animal models of a wide range of tumour types including breast, prostate and colon, as well as some leukemias.
Lexibulin is an orally bioavailable small-molecule with tubulin-inhibiting, vascular-disrupting, and potential antineoplastic activities. Lexibulin inhibits tubulin polymerization in tumor blood vessel endothelial cells and tumor cells, blocking the formation of the mitotic spindle and leading to cell cycle arrest at the G2/M phase; this may result in disruption of the tumor vasculature and tumor blood flow, and tumor cell death.

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Drug Indication
Investigated for use/treatment in solid tumors.

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Mechanism of Action
CYT997 is a vascular disrupting agent and a tubulin inhibitor for various cancers. The essential role of microtubules in cell division and the capacity of drugs that interact with the protein subunits of microtubules (α- and β-tubulin) to interfere with the cell cycle, have made tubulin a highly successful target for the development of therapeutic drugs, such as anti-cancer drugs and vascular disrupting agents.

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Lexibulin (CYT-997) is a novel orally active small-molecule inhibitor with dual activities: tubulin polymerization inhibition and vascular disrupting effects [1]
Its antitumor mechanisms include: inhibiting microtubule dynamics to induce G2/M arrest and apoptosis; disrupting tumor vasculature to block nutrient supply; inducing mitochondrial ROS accumulation to suppress JAK2/STAT3 pathway, thereby promoting autophagy and apoptosis [1][2][3]
It exhibits broad-spectrum antiproliferative activity against solid tumors (lung, colon, gastric, breast cancer) and hematological malignancies (multiple myeloma) [1][2][3]
As an oral agent with favorable pharmacokinetic properties and low toxicity, it has potential clinical applications for the treatment of various cancers [1]
Its unique combination of cell cytotoxicity and vascular disruption distinguishes it from traditional tubulin inhibitors, enhancing antitumor efficacy [1]

C24H30N6O2

434.53

434.243

917111-44-5

Lexibulin dihydrochloride;917111-49-0

11351021

White to off-white solid powder

1.2±0.1 g/cm3

557.7±50.0 °C at 760 mmHg

291.1±30.1 °C

0.0±1.5 mmHg at 25°C

1.610

4.14

107.78

3

6

9

32

565

1

CCC[C@@H](C1=CN=CC=C1)NC2=NC(=NC=C2C)C3=CC(=C(C=C3)NC(=O)NCC)OC

MTJHLONVHHPNSI-IBGZPJMESA-N

InChI=1S/C24H30N6O2/c1-5-8-19(18-9-7-12-25-15-18)28-22-16(3)14-27-23(30-22)17-10-11-20(21(13-17)32-4)29-24(31)26-6-2/h7,9-15,19H,5-6,8H2,1-4H3,(H2,26,29,31)(H,27,28,30)/t19-/m0/s1

(S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2-yl)phenyl)urea

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: ≥ 2.5 mg/mL (5.75 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 (5.75 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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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。