p53 (IC50 = 0.37 μM); NF-κB (IC50 = 0.47 μM); FACT
FACT (facilitates chromatin transcription) complex [1]
FACT (facilitates chromatin transcription) complex [2]

CBL0137 是胰腺癌细胞系细胞凋亡的有效诱导剂，对胰腺癌干细胞和活跃增殖的肿瘤细胞有毒。使用 CBL0137 和相关分子可以抑制 NF-B 和 HSF-1 控制的细胞应激途径，同时激活 p53 [2]。 CBL0137 结合 DNA 时不会引入任何类型的化学改变，使其无基因毒性。促进染色质转录 (FACT) 复合物是一种参与转录、复制和 DNA 修复的染色质重塑复合物，由于 CBL0137 与 DNA 结合而功能失活。在 CBL0137 处理的细胞中，FACT 从核质中丢失并被捕获在染色质中，从而抑制 FACT 依赖性转录，包括 NF-kB 介导的转录。此外，FACT 的染色质捕获导致 p53 以酪蛋白激酶 2 (CK2) 依赖性方式磷酸化和激活 [3]。

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CBL0137盐酸盐（CBLC137）对人纤维肉瘤HT1080细胞具有细胞毒性（具有特定IC₅₀值），对正常二倍体成纤维细胞（Wi38、MEF）的毒性较低（P < 0.001）。用2 μM CBLC137处理24小时可诱导肿瘤细胞（HT1080、RCC45、MiaPaca）细胞周期停滞，但对正常细胞（Wi38、NKE-hTERT）影响较小。它通过酪蛋白激酶2（CK2）促进p53 Ser³⁹²磷酸化来激活p53，并通过将FACT捕获在染色质中抑制NF-κB依赖性转录，且不会诱导可检测到的DNA损伤（彗星实验和H2AX磷酸化实验证实无DNA断裂）。此外，它还会降低FACT亚基（SSRP1、SPT16）的可溶性部分，阻断NF-κB的核质穿梭和DNA结合 [1]
CBL0137盐酸盐（CBLC137）是胰腺癌细胞系（MiaPaCa-2、PANC-1、BxPC-3）的强效凋亡诱导剂，用2 μM处理4-24小时后，可检测到半胱天冬酶3、7、8、9和PARP1的切割。它能抑制胰腺癌细胞和耐药癌干细胞（CSCs）的活力，降低CSC表面标志物（CD24ᵂⁱ CD44ᵂⁱ）的比例，阻止吉西他滨诱导的“侧群”富集。在集落形成实验（MiaPaCa-2、PANC-1）中与吉西他滨表现出协同作用，并能抑制吉西他滨诱导的转录反应，包括NF-κB靶基因表达和核糖核苷酸还原酶（RNR）的RRM1/RRM2亚基 [2]

在小鼠中，CBL0137 可有效对抗多种胰腺导管腺癌 (PDA) 模型，包括原位吉西他滨耐药 PANC-1 模型和患者来源的异种移植物，其中 CBL0137 的抗肿瘤作用与 FACT 的过度表达相关[1]。根据其提出的作用机制，CBL0137靶向胶质母细胞瘤（GBM），穿透血脑屏障，并且在TMZ反应性和耐药性原位模型中均有效。这种药物能够穿过血脑屏障，尤其是静脉注射时，其治疗中枢神经系统肿瘤的潜力令人鼓舞。在原位模型中，静脉注射药物比口服药物具有更高的生物利用度，因为它们在肿瘤组织中积累更多。 CBL0137 在脑组织中的正常积累不会导致可检测到的神经毒性[3]。

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CBL0137盐酸盐（CBLC137）在多种人类肿瘤异种移植小鼠模型中表现出抗肿瘤活性。对于肾细胞癌Caki-1、结直肠癌DLD-1、黑色素瘤Mel-7和胰腺导管腺癌（PDA），CBLC137处理与溶媒组相比显著降低肿瘤体积（P < 0.005，方差分析），每组5-10只小鼠。在患有自发性乳腺肿瘤的MMTV-neu转基因小鼠中，口服灌胃100 mg/kg CBLC137后24小时，肿瘤中SSRP1和SPT16的可溶性部分减少（P < 0.05） [1]
CBL0137盐酸盐（CBLC137）在胰腺癌的原位和患者来源异种移植（PDX）模型中显示出疗效。在原位吉西他滨耐药PANC-1模型（每组6-7只）中，每周静脉注射90 mg/kg CBLC137，持续4周，可减少肿瘤体积，与吉西他滨（40 mg/kg腹腔注射，每4天一次）联合使用可增强抗肿瘤效果。在PDX模型（PDX#13756、#13590；每组5-10只）中，50-90 mg/kg CBLC137静脉注射每周一次（单独或与20 mg/kg吉西他滨腹腔注射每4天一次联合使用），持续4周，可抑制肿瘤生长，其抗肿瘤效果与FACT过表达相关 [2]

将 CBL0137 盐酸盐应用于 MiaPaca2 和 BxPC-3 细胞 4 或 24 小时。蛋白酶和磷酸酶抑制剂存在于 1× 细胞培养裂解试剂中，用于收获细胞。在 SDS-PAGE 凝胶上分离后，将 5 至 20 μg 裂解物转移到 PVDF 膜上。靶向 SSRP1、SPT16、RRM1 和 RRM2 蛋白的抗体用于探测印迹。上样对照是 GAPDH。 ECL 试剂盒用于可视化蛋白质[1]。

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从经CBL0137盐酸盐（CBLC137）、奎纳克林或紫外线处理的HT1080细胞裂解物中免疫沉淀CK2β，进行CK2激酶活性测定。使用对应于p53 C末端（311-393位氨基酸）的肽底物，检测药物诱导的CK2介导的磷酸化 [1]

将细胞重悬于无血清 Dulbecco's Modified Eagle Medium (DMEM) 中，并暴露于不同浓度的 CBL0137 盐酸盐中 1 小时。然后，将来自每种处理条件的 105 个细胞接种到 6 孔板的 3 个孔中，加入 2 mL 无血清 DMEM/F12 培养基，补充有 0.4% BSA、0.2×B27、10 ng/mL 重组 EGF，并含有 0.25 ％琼脂糖。在具有常规含 FBS 培养基的 6 孔板的三个孔中，将来自每种处理条件的 103 个细胞铺板。平板接种后七至十五天，在倒置显微镜下对菌落进行计数。

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细胞毒性实验：将肿瘤细胞（HT1080、RCC45、MiaPaca）和正常细胞（Wi38、NKE-hTERT）用不同浓度的CBL0137盐酸盐（CBLC137）处理24-72小时，通过Cell Titer Blue实验或亚甲基蓝染色检测细胞活力。细胞周期分析：用2 μM CBLC137处理细胞24小时后，用碘化丙啶染色，通过流式细胞术（FACS）分析。蛋白质印迹法：用CBLC137（0.8-2 μM）处理HT1080细胞8-16小时，检测p53 Ser³⁹²磷酸化、FACT亚基表达以及半胱天冬酶/PARP1切割。RT-PCR/qPCR：用1-2 μM CBLC137处理HT1080或MiaPaCa-2细胞2-24小时，定量NF-κB靶基因（IL-8、TNF、IκBα）和RNR亚基（RRM1、RRM2）的mRNA表达。染色质免疫沉淀（ChIP）：用1-2 μM CBLC137处理HT1080细胞2小时（有/无TNF刺激），分析SSRP1、p65和RNA聚合酶II与NF-κB依赖性基因启动子（IL-8、TNF）的结合情况。CSC相关实验：用3 μM CBLC137处理PANC-1细胞1小时，用Hoechst 33342或CSC表面标志物（CD24、CD44）染色，流式细胞术分析；药物处理后计数2D/3D培养基中的集落形成情况 [1][2]

Ketamine/xylazine is used to create a deep anesthesia in 10-week-old female athymic nude mice (n = 8 per treatment group). Each mouse has its pancreas tail inoculated with 2 106 PANC-1 cells via laparotomy. When the tumor was detected by ultrasound two weeks after the vaccination, treatment started. There are the following routines: 1) Vehicles, gavage of sterile water and 100 mg/kg captisol 2) 100 mg/mL captisol diluted with 50 to 90 mg/kg CBL0137 hydrochloride and given intravenously once a week via the tail vein; 3) 10 to 20 mg/kg CBL0137 hydrochloride given orally five days on and two days off. To measure tumors, digital calipers are used. The equation LW2/2 is used to determine the tumor volume, where L is the longest dimension and W is the dimension that is perpendicular to W. Mice are monitored for at least 90 days after the start of treatment or until at least one tumor per mouse reached 1000 mm3, whichever occurs first.

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Xenograft tumor models: Human tumor cells (Caki-1, DLD-1, Mel-7, PDA, PANC-1) or PDX tissues were inoculated into nude mice or SCID mice. When tumors reached the specified volume, CBL0137 HCl (CBLC137) was administered intravenously at 50-90 mg/kg once weekly, alone or combined with gemcitabine (20-40 mg/kg i.p. every 4 days) for 4 weeks. Tumor volume was measured periodically, and mice were euthanized 1 week post-treatment for tumor collection and analysis. Spontaneous tumor model: MMTV-neu transgenic mice with palpable mammary tumors were given 100 mg/kg CBLC137 via oral gavage, and tumor lysates were collected 24 hours later to detect soluble FACT subunits by Western blot [1][2]

CBL0137 HCl (CBLC137) showed selective toxicity to tumor cells, with lower IC₅₀ values for tumor cells (HT1080, C8) than normal diploid fibroblasts (Wi38, MEF). It did not induce detectable DNA damage in HeLa or HT1080 cells, as confirmed by comet assay (no increase in tail moment) and lack of histone H2AX phosphorylation [1]

[1]. Sci Transl Med . 2011 Aug 10;3(95):95ra74.

[2]. Oncotarget . 2014 Nov 30;5(22):11038-53.

[3]. Neuro Oncol . 2017 Feb 1;19(2):186-196.

Effective eradication of cancer requires treatment directed against multiple targets. The p53 and nuclear factor κB (NF-κB) pathways are dysregulated in nearly all tumors, making them attractive targets for therapeutic activation and inhibition, respectively. We have isolated and structurally optimized small molecules, curaxins, that simultaneously activate p53 and inhibit NF-κB without causing detectable genotoxicity. Curaxins demonstrated anticancer activity against all tested human tumor xenografts grown in mice. We report here that the effects of curaxins on p53 and NF-κB, as well as their toxicity to cancer cells, result from \"chromatin trapping\" of the FACT (facilitates chromatin transcription) complex. This FACT inaccessibility leads to phosphorylation of the p53 Ser(392) by casein kinase 2 and inhibition of NF-κB-dependent transcription, which requires FACT activity at the elongation stage. These results identify FACT as a prospective anticancer target enabling simultaneous modulation of several pathways frequently dysregulated in cancer without induction of DNA damage. Curaxins have the potential to be developed into effective and safe anticancer drugs.[1]

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\n\nPancreatic ductal adenocarcinoma (PDA) continues to be one of the deadliest cancers due to the absence of effective treatment. Curaxins are a class of small molecules with anti-cancer activity demonstrated in different models of cancer in mice. The lead curaxin compound, CBL0137, recently entered Phase I clinical trials. Curaxins modulate several important signaling pathways involved in the pathogenesis of PDA through inhibition of chromatin remodeling complex FACT. FACT is overexpressed in multiple types of tumor, with one of the highest rate of overexpression in PDA (59%). In this study, the efficacy of CBL0137 alone or in combination with current standard of care, gemcitabine, was tested against different models of PDA in vitro and in mouse models. It was found that CBL0137 alone is a potent inducer of apoptosis in pancreatic cancer cell lines and is toxic not only for proliferating bulk tumor cells, but also for pancreatic cancer stem cells. In mice, CBL0137 was effective against several PDA models, including orthotopic gemcitabine resistant PANC-1 model and patient derived xenografts, in which CBL0137 anti-tumor effect correlated with overexpression of FACT. Moreover, we observed synergy of CBL0137 with gemcitabine which may be explained by the ability of CBL0137 to inhibit several transcriptional programs induced by gemcitabine, including NF-kappaB response and expression of ribonucleotide reductase, one of the targets of gemcitabine in cells. This data suggest testing of CBL0137 efficacy in Phase II trial in PDA patients alone and in combination with gemcitabine.[2]

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\n\nPancreatic ductal adenocarcinoma (PDA) continues to be one of the deadliest cancers due to the absence of effective treatment. Curaxins are a class of small molecules with anti-cancer activity demonstrated in different models of cancer in mice. The lead curaxin compound, CBL0137, recently entered Phase I clinical trials. Curaxins modulate several important signaling pathways involved in the pathogenesis of PDA through inhibition of chromatin remodeling complex FACT. FACT is overexpressed in multiple types of tumor, with one of the highest rate of overexpression in PDA (59%). In this study, the efficacy of CBL0137 alone or in combination with current standard of care, gemcitabine, was tested against different models of PDA in vitro and in mouse models. It was found that CBL0137 alone is a potent inducer of apoptosis in pancreatic cancer cell lines and is toxic not only for proliferating bulk tumor cells, but also for pancreatic cancer stem cells. In mice, CBL0137 was effective against several PDA models, including orthotopic gemcitabine resistant PANC-1 model and patient derived xenografts, in which CBL0137 anti-tumor effect correlated with overexpression of FACT. Moreover, we observed synergy of CBL0137 with gemcitabine which may be explained by the ability of CBL0137 to inhibit several transcriptional programs induced by gemcitabine, including NF-kappaB response and expression of ribonucleotide reductase, one of the targets of gemcitabine in cells. This data suggest testing of CBL0137 efficacy in Phase II trial in PDA patients alone and in combination with gemcitabine.[3]

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CBL0137 HCl (CBLC137) is a lead curaxin compound, a class of small-molecule anticancer agents that target the FACT complex [1][2]
Its mechanism of action involves "chromatin trapping" of FACT, leading to p53 activation (via CK2-mediated Ser³⁹² phosphorylation) and NF-κB inhibition (blocking transcription elongation), without causing genotoxicity [1]
FACT is overexpressed in multiple tumors (59% overexpression rate in pancreatic ductal adenocarcinoma), and CBL0137’s antitumor effect correlates with FACT expression levels [2]
It eradicates drug-resistant cancer stem cells and potentiates gemcitabine efficacy by inhibiting gemcitabine-induced NF-κB response and RNR expression [2]
CBL0137 has entered Phase I clinical trials and is proposed for Phase II trials in pancreatic cancer patients (alone or combined with gemcitabine) [2]

C21H24N2O2-HCL

372.89

372.16

C, 67.64; H, 6.76; Cl, 9.51; N, 7.51; O, 8.58

1197397-89-9

CBL0137;1197996-80-7

44519123

Off-white to yellow solid powder

5.39

51.1

2

3

6

26

466

0

CC(C1C=C2C(N(CCNC(C)C)C3=CC=C(C=C32)C(C)=O)=CC=1)=O.Cl

IXRKBBVMDMKAEB-UHFFFAOYSA-N

InChI=1S/C21H24N2O2.ClH/c1-13(2)22-9-10-23-20-7-5-16(14(3)24)11-18(20)19-12-17(15(4)25)6-8-21(19)23;/h5-8,11-13,22H,9-10H2,1-4H3;1H

1-[6-acetyl-9-[2-(propan-2-ylamino)ethyl]carbazol-3-yl]ethanone;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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 (6.70 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 (6.70 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 25.0 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: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 0.75mg/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：
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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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