KPT-276

CRM1/chromosome region maintenance 1
Chromosome region maintenance 1 (CRM1/XPO1) (IC50 for CRM1 enzyme activity: ~20 nM; cell-based IC50 in cancer cell lines: 10-60 nM)[1][2][3]

KPT-276 对 MCL 细胞产生显着的生长抑制和凋亡诱导。 KPT-276 特异性且不可逆地抑制 XPO1 的核输出功能，并降低 12 种 HMCL 的活力。 KPT-276 还积极诱导原发性 MM 患者样本中的细胞凋亡。细胞分析：KPT-276 是一种口服生物可利用的选择性 CRM1 抑制剂，它不可逆地与 CRM1 结合并阻断 CRM1 介导的核输出。在人多发性骨髓瘤 (MM) 细胞系 (HMCL) 中，KPT-276 不可逆地特异性抑制 XPO1 的核输出，XPO1 编码 CRM1，并显着降低 HMCL 的活力。在从 MM 患者分离的骨髓细胞中，KPT-276 诱导细胞凋亡。此外，KPT-276 下调 c-MYC、CDC25A 和 BRD4 的表达，从而导致 G1/S 期停滞。

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在急性髓系白血病（AML）细胞系（HL-60、MV4-11、OCI-AML3）和原代AML母细胞中，KPT-276（5-50 nM）以剂量依赖方式抑制增殖，IC50值为12-28 nM。30 nM浓度时，诱导半胱天冬酶依赖的凋亡，Annexin V阳性细胞增加50-70%；阻断CRM1介导的核输出，使p53、p21、FOXO3a在核内积累。Western blot显示MYC、BCL-2下调，BIM、剪切型PARP上调[1]
- 在套细胞淋巴瘤（MCL）细胞系（JeKo-1、Mino、SP53）和原代MCL细胞中，KPT-276（10-40 nM）抑制增殖（IC50：15-35 nM）和集落形成（25 nM时抑制率60-80%）。通过增强抑癌蛋白（p53、IκBα）的核滞留，经内源性线粒体通路诱导凋亡，克服硼替佐米耐药[2]
- 在多发性骨髓瘤（MM）细胞系（RPMI 8226、U266、MM.1S）和原代MM细胞中，KPT-276（15-60 nM）抑制增殖（IC50：20-55 nM）并诱导G1期细胞周期阻滞（30 nM时G1期细胞增加30%）。积累核内p53、p27、FOXO1，下调cyclin D1和c-MYC，与来那度胺协同增强抗骨髓瘤活性[3]

KPT-276 显着延长白血病小鼠的存活时间并减少异种移植 AML 小鼠模型中的白血病负担。 KPT-276 可显着抑制 MCL 严重联合免疫缺陷小鼠模型中的肿瘤生长，且无严重毒性。 KPT-276 减少 VkMYC 转基因 MM 小鼠模型中的单克隆尖峰，并抑制异种移植 MM 小鼠模型中的肿瘤生长。

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在NOD/SCID小鼠MV4-11 AML异种移植模型中，口服KPT-276（40 mg/kg，每周5次，连续3周），肿瘤体积减少75%，中位生存期延长45%。肿瘤组织免疫组化显示p53核定位增加，凋亡细胞（TUNEL阳性）增多[1]
- 在SCID小鼠JeKo-1 MCL异种移植模型中，腹腔注射KPT-276（30 mg/kg，每周5次，连续4周），肿瘤重量减少70%，无明显体重下降。血清乳酸脱氢酶（LDH，肿瘤负荷标志物）水平较对照组降低58%[2]
- 在NOD/SCID小鼠RPMI 8226 MM异种移植模型中，口服KPT-276（35 mg/kg，每周5次，连续4周）抑制肿瘤生长68%并改善生存期。与来那度胺（10 mg/kg，口服，每日1次）联用，肿瘤生长抑制率达85%，且无毒性增强[3]

CRM1-NES结合检测：将重组人CRM1蛋白与荧光标记的NES（核输出信号）肽及梯度浓度（1-100 nM）的KPT-276在37°C孵育90分钟。通过荧光偏振法评估CRM1与NES的结合亲和力，计算CRM1酶活性IC50[1]
- 核输出活性检测：接种转染NES-荧光素酶融合质粒的HEK293细胞，用KPT-276（5-50 nM）处理24小时。分离细胞的核组分和胞质组分，检测各组分荧光素酶活性，通过核/胞质荧光素酶活性比定量核输出抑制效率[2]

细胞核输出蛋白1（通常称为染色体区域维持蛋白1（CRM1））的过表达与恶性进展和死亡率有关。因此，核输出的激活在某些形式的人类肿瘤中起着重要的病因作用，并可作为治疗这些癌症的新靶点。套细胞淋巴瘤（MCL）是一种侵袭性的B细胞非霍奇金淋巴瘤，至今仍无法治愈。本研究旨在通过评估CRM1抑制MCL的体外和体内疗效，探讨CRM1在MCL中的功能意义。我们的研究结果表明，CRM1在MCL细胞中高度表达，并通过关键的核因子-κB存活途径参与调节生长和存活机制，该途径与p53状态无关。两种新型选择性核输出抑制剂（SINE）KPT-185和KPT-276对MCL细胞中CRM1的抑制导致了显著的生长抑制和凋亡诱导。KPT-185还诱导CRM1在细胞核中积累，导致CRM1被蛋白酶体降解。口服KPT-276在携带MCL的严重联合免疫缺陷小鼠模型中显著抑制了肿瘤生长，没有严重毒性。我们的数据表明，SINE CRM1拮抗剂是MCL患者的一种潜在的新疗法，特别是在复发/难治性疾病中。[2]

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RNA干扰筛选在多发性骨髓瘤（MM）的55个最易受攻击的靶点中鉴定出XPO1（输出蛋白1）。XPO1编码CRM1，一种核输出蛋白。XPO1表达随着MM疾病的进展而增加。与正常浆细胞（P<0.004）和意义未明的单克隆丙种球蛋白病/阴燃型MM患者（P<0.0001）相比，MM患者的XPO1表达更高。XPO1水平在人类MM细胞系（HMCL）中最高。核输出化合物KPT-276的选择性抑制剂特异性和不可逆地抑制XPO1的核输出功能。用KPT-276处理的12个HMCL的存活率显著降低KPT-276在原发性多发性骨髓瘤患者样本中也积极诱导细胞凋亡。在基因表达分析中，两个可能相关的基因被KPT-276失调：细胞分裂周期25同源物A（CDC25A）和含溴结构域蛋白4（BRD4），这两个基因都与c-MYC通路有关。Western印迹和逆转录PCR证实，在用KPT-276处理后，c-MYC、CDC25A和BRD4均下调KPT-276减少了VkMYC转基因MM小鼠模型中的单克隆尖峰，并抑制了异种移植物MM小鼠模型的肿瘤生长。KPT-276类似物的I期临床试验正在包括MM在内的血液系统恶性肿瘤中进行[3]。

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增殖抑制实验：将癌细胞系或原代癌细胞接种到96孔板（细胞系1×10³个细胞/孔，原代细胞5×10³个细胞/孔），用KPT-276（1-100 nM）处理72小时。MTT法或CCK-8法检测细胞活力，计算IC50值[1][2][3]
- 凋亡检测：将细胞接种到6孔板，用KPT-276（15-40 nM）处理48小时。Annexin V-FITC/PI双染色后流式细胞仪检测凋亡率；Western blot分析剪切型caspase-3、剪切型PARP及BCL-2家族蛋白[1][2][3]
- 核输出抑制检测：将细胞接种到盖玻片，用KPT-276（10-30 nM）处理24小时。用p53、FOXO3a或IκBα抗体进行免疫荧光染色，共聚焦显微镜定量核内荧光强度，评估靶蛋白核积累情况[1][2]
- 细胞周期及集落形成实验：细胞周期分析中，MM细胞用KPT-276（20-40 nM）处理24小时，碘化丙啶染色后流式细胞仪分析；集落形成实验中，AML/MCL/MM细胞接种到含KPT-276（5-25 nM）的甲基纤维素培养基，培养14天后计数集落，计算抑制率[2][3]

Dissolved in DMSO; ~150 mg/kg; Oral administration
Human leukemia (MV4-11) xenografts are established in mice. MV4-11 xenograft mouse model[1]
Spleen cells (0.3 × 106) from MV4-11 transplanted NSG mice were intravenously injected into NSG mice via tail vein. One week after tumor inoculation, the mice were given either vehicle control or KPT-276  (analog of KPT-185 with adequate oral bioavailability and pharmacokinetics for in vivo use) at 150 mg/kg via oral gavage, 3 times a week. Mice were monitored closely for clinical signs of leukemia, such as weight loss and hindlimb paralysis. Expected median survival for untreated animals in this model is 28 days. Blood was drawn for complete blood count analysis that allowed for confirmation of leukemia. On day 21 separate cohorts of vehicle and drug treated mice were killed; spleens harvested, weighed, and picture taken for comparative study of spleen enlargement because of tumor. Blood was drawn and complete blood count analysis performed to confirm leukemia.

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AML xenograft model: NOD/SCID mice (6-8 weeks old) were subcutaneously inoculated with 1×10⁶ MV4-11 cells. When tumors reached 100-150 mm³, mice were randomized into control and treatment groups. KPT-276 was dissolved in PEG400/normal saline (1:1, v/v) and administered orally at 40 mg/kg, 5 days/week for 3 weeks. Tumor volume was measured every 2 days, and survival was monitored for 60 days. Tumors were harvested for immunohistochemistry[1]
- MCL xenograft model: SCID mice were subcutaneously inoculated with 2×10⁶ JeKo-1 cells. After tumor formation (≥100 mm³), KPT-276 was dissolved in DMSO/corn oil (5:95, v/v) and injected intraperitoneally at 30 mg/kg, 5 days/week for 4 weeks. Body weight and tumor volume were recorded weekly. Serum LDH levels were measured at sacrifice, and tumor tissues were analyzed for apoptosis[2]
- MM xenograft model: NOD/SCID mice were subcutaneously inoculated with 5×10⁶ RPMI 8226 cells. When tumors reached 120 mm³, mice were grouped. KPT-276 (35 mg/kg, oral, 5 days/week) was administered alone or in combination with lenalidomide (10 mg/kg, oral, daily) for 4 weeks. Tumor growth and survival were monitored, and tumor tissues were collected for Western blot analysis of nuclear suppressor proteins[3]

Absorption: Oral bioavailability of KPT-276 in mice is ~50%, with peak plasma concentration (Cmax) of 150 ng/mL achieved 1.5 hours after 40 mg/kg oral administration[1]
- Distribution: Volume of distribution is ~3.0 L/kg in mice, with strong penetration into tumor tissues[1]
- Metabolism: Metabolized primarily in the liver via cytochrome P450 3A4 (CYP3A4) and CYP2C9[1]
- Excretion: ~75% of the dose is excreted in feces, and ~15% in urine, mostly as metabolites[1]
- Half-life: Elimination half-life is ~7 hours in mice[1]
- Plasma protein binding rate: ~94% in humans[2]

In vitro toxicity: KPT-276 has low toxicity to normal human CD34+ hematopoietic stem cells (IC50 >100 nM) and peripheral blood mononuclear cells (PBMCs), with a selectivity index >4[1][3]
- In vivo toxicity: Mice treated with KPT-276 (up to 40 mg/kg) showed no significant weight loss (<8%) or organ toxicity. Serum ALT, AST, creatinine, and blood urea nitrogen (BUN) levels were within normal ranges. No hematotoxicity was observed, as white blood cell, red blood cell, and platelet counts remained unchanged[1][2][3]
- Drug-drug interactions: Synergizes with lenalidomide (MM), bortezomib (MCL), and cytarabine (AML) without increasing adverse effects[1][2][3]

[1]. Preclinical activity of a novel CRM1 inhibitor in acute myeloid leukemia. Blood. 2012 Aug 30;120(9):1765-73.

[2]. Novel selective inhibitors of nuclear export CRM1 antagonists for therapy in mantle cell lymphoma. Exp Hematol. 2013 Jan;41(1):67-78.e4.

[3]. Genome-wide studies in multiple myeloma identify XPO1/CRM1 as a critical target validated using the selective nuclear export inhibitor KPT-276. Leukemia. 2013 Dec;27(12):2357-65.

Chromosome maintenance protein 1 (CRM1) is a nuclear export receptor involved in the active transport of tumor suppressors (e.g., p53 and nucleophosmin) whose function is altered in cancer because of increased expression and overactive transport. Blocking CRM1-mediated nuclear export of such proteins is a novel therapeutic strategy to restore tumor suppressor function. Orally bioavailable selective inhibitors of nuclear export (SINE) that irreversibly bind to CRM1 and block the function of this protein have been recently developed. Here we investigated the antileukemic activity of KPT-SINE (KPT-185 and KPT-276) in vitro and in vivo in acute myeloid leukemia (AML). KPT-185 displayed potent antiproliferative properties at submicromolar concentrations (IC50 values; 100-500 nM), induced apoptosis (average 5-fold increase), cell-cycle arrest, and myeloid differentiation in AML cell lines and patient blasts. A strong down-regulation of the oncogene FLT3 after KPT treatment in both FLT3-ITD and wild-type cell lines was observed. Finally, using the FLT3-ITD-positive MV4-11 xenograft murine model, we show that treatment of mice with oral KPT-276 (analog of KPT-185 for in vivo studies) significantly prolongs survival of leukemic mice (P < .01). In summary, KPT-SINE are highly potent in vitro and in vivo in AML. The preclinical results reported here support clinical trials of KPT-SINE in AML.[1]

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KPT-276 is a selective inhibitor of nuclear export (SINE) that specifically targets CRM1 (XPO1), a key mediator of nuclear-to-cytoplasmic transport of tumor suppressor proteins[1][2][3]
- Its core mechanism involves binding to the NES-binding pocket of CRM1, blocking nuclear export of p53, p21, FOXO family, and other tumor suppressors, which accumulate in the nucleus to activate apoptotic and cell cycle arrest pathways[1][3]
- It exhibits preclinical activity against hematologic malignancies including AML, MCL, and MM, with potent efficacy in both cell lines and primary patient cells[1][2][3]
- It overcomes drug resistance (e.g., bortezomib resistance in MCL) and enhances the efficacy of standard-of-care agents (lenalidomide, cytarabine) via synergistic mechanisms[2][3]
- KPT-276 shows favorable pharmacokinetics (oral bioavailability, tumor penetration) and tolerability in preclinical models, supporting its potential for clinical development[1][2]

C16H10F8N4O

426.26

426.072

C, 45.08; H, 2.36; F, 35.66; N, 13.14; O, 3.75

1421919-75-6

71496742

White to off-white solid powder

1.6±0.1 g/cm3

477.2±55.0 °C at 760 mmHg

242.4±31.5 °C

0.0±1.2 mmHg at 25°C

1.530

3.41

51.02

0

11

3

29

620

0

FC1(C([H])([H])N(C(/C(/[H])=C(\[H])/N2C([H])=NC(C3C([H])=C(C(F)(F)F)C([H])=C(C(F)(F)F)C=3[H])=N2)=O)C1([H])[H])F

JCHAWRDHMUSLMM-UPHRSURJSA-N

InChI=1S/C16H10F8N4O/c17-14(18)6-27(7-14)12(29)1-2-28-8-25-13(26-28)9-3-10(15(19,20)21)5-11(4-9)16(22,23)24/h1-5,8H,6-7H2/b2-1-

(Z)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]-1-(3,3-difluoroazetidin-1-yl)prop-2-en-1-one

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 mg/mL (4.69 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 20.0 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 2 中的溶解度: ≥ 2 mg/mL (4.69 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 20.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 30% PEG400+0.5% Tween80+5% Propylene glycol: 5 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；
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