H3B-8800

SF3B splicing

H3B-8800有效且优先杀死剪接体突变的上皮和血液肿瘤细胞。H3B-8800的这些杀伤作用是由于其与SF3b复合物的直接相互作用，如在编码SF3b成分的基因突变的耐药细胞中H3B-8800的活性损失所证明的。

每天口服2或4 mg H3B-8800/kg体重（mg/kg）可减缓SF3B1K700E异种移植物的生长（P＜0.003，双向方差分析，然后进行Dunnett多重比较试验），但对SF3B1WT异种移植物没有影响。尽管8 mg/kg的剂量确实减缓了SF3B1WT肿瘤的生长，但它完全消除了SF3b1K000E肿瘤的生长（P＜0.003）。b）。H3B-8800同样对携带编码SF3B1-K700E的内源性突变的人HNT-34急性髓系白血病（AML）细胞的异种移植物显示出显著的抗肿瘤活性。H3B-8800在携带SF3B1WT或SF3B1K700E异种移植物的小鼠的血浆和肿瘤中达到相似的浓度，并且H3B-8800以剂量依赖的方式调节规范剪接和异常剪接。根据H3B-8800的药代动力学特征，早在治疗后1小时就观察到剪接调节，并且在治疗后24小时内剪接恢复到预处理水平。

从过表达Flag标记的SF3B1的293F细胞制备的核提取物中免疫沉淀SF3B复合物。首先，通过孵育80μg抗SF3B1抗体（MBL International D221-3，anti-Sap155单克隆抗体）和24 mg抗小鼠PVT闪烁邻近测定（SPA）珠（PerkinElmer）30分钟，将抗体批量固定在珠上。离心（14000 r.p.m.，在4°C下5分钟）后，将抗体-珠混合物重悬于补充有PhosSTOP磷酸酶抑制剂混合物（Roche）和cOmplete ULTRA蛋白酶抑制剂混合物（罗氏）的PBS中。通过用磷酸酶和蛋白酶抑制剂将40 mg稀释到总体积为16 ml的PBS中制备核提取物，并将混合物离心（14000 r.p.m.，在4°C下离心10分钟）。将上清液转移到干净的管中，加入抗体-珠混合物并孵育2小时。通过离心收集珠，用PBS+0.1%Triton X-100洗涤两次，并用4.8ml PBS重悬。使用浆液和不同浓度的H3B-8800制备100μl结合反应。在室温下预培养15分钟后，加入Kotake等人20中使用的1nM 3H探针。将混合物在室温下孵育15分钟，并使用MicroBeta2平板计数器（PerkinElmer）读取发光信号。

胰腺癌症细胞以每孔750个细胞接种在384孔板中，并在第2天用化合物处理。将K562等基因细胞（K562-SF3B1K700E和K562-SF3 B1K700K）以每孔10000个细胞接种在96孔板中，4小时后用化合物处理。在指示的时间点使用CellTiter-Glo或Caspase-Glo 3/7（Promega）通过发光测量活细胞和凋亡细胞的相对数量。

K562 isogenic lines, HNT-34 and K052 xenograft models efficacy and pharmacokinetic/pharmacodynamic modeling. 1 × 107 K562 isogenic, HNT-34, or K052 cells were subcutaneously implanted into the flank of female NSG or CB17-SCID mice of 6–8 weeks of age. Mice were treated with H3B-8800 (10% ethanol, 5% Tween-80, 85% saline) or vehicle control. For the efficacy studies, the mice were orally dosed daily, and the mice were monitored until they reached either of the following endpoints: (i) excessive tumor volume (≥20 mm in its longest diameter), which was measured three times a week (tumor volume calculated by using the ellipsoid formula: (length × width2) / 2), or (ii) development of any health problem, such as paralysis or excessive body weight loss. The differences in tumor volume during the study period between the vehicle-treated and H3B-8800-treated groups were analyzed by two-way analysis of variance (ANOVA) followed by the Dunnett’s multiple comparison test. For the pharmacokinetic/pharmacodynamic modeling (PK/PD) studies, the mice were treated with one dose, and the tumors were collected at the indicated times (in Figs. 2 and ​and3)3) after treatment for further analysis. RNA was isolated using RiboPure RNA purification kit (Ambion) and used for TLDA. All mouse studies were carried out under protocols approved by the Institutional Animal Care and Use Committee at H3 Biomedicine.

[1]. H3B-8800, an orally available small-molecule splicing modulator, induces lethality in spliceosome-mutant cancers. Nat Med. 2018 May;24(4):497-504.

H3B-8800 is a novel spliceosome inhibitor developed by H3 Biomedicine. It offers the benefit of preferentially killing spliceosome-mutant cancer cells whereas other splicesome inhibitors, such as the pladienolide analogue E7107, show no such preferential targeting. H3B-8800 was granted orphan drug status by the FDA in August 2017 and is in clinical trials for the treatment of acute myelogenous leukemia and chronic myelomonocytic leukemia.
Splicing Inhibitor H3B-8800 is an orally bioavailable inhibitor of the splicing factor 3B subunit 1 (SF3B1), with potential antineoplastic activity. Upon administration, H3B-8800 binds to and blocks the activity of SF3B1, a core spliceosome protein that is mutated in various cancer cells. This modulates RNA splicing by preventing aberrant mRNA splicing by the spliceosome, blocks RNA mis-splicing, enhances proper RNA splicing and prevents the expression of certain tumor-associated genes. This leads to an induction of apoptosis and prevents tumor cell proliferation. In many cancer cells, core spliceosome proteins, including SF3B1, U2 small nuclear ribonucleoprotein auxiliary factor 1 (U2AF1), serine/arginine-rich splicing factor 2 (SRSF2) and U2 small nuclear ribonucleoprotein auxiliary factor subunit-related protein 2 (ZRSR2), are mutated and aberrantly activated leading to a dysregulation of mRNA splicing.

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Mechanism of Action
H3B-8800 is thought to bind to a site similar to pladienolides on the SF3B complex within the spliceosome. Once bound it induces increased retention of short (<300 nucleotide) GC-rich introns through modulation of pre-mRNA processing. These intron-retained mRNA sequences are then thought to be destroyed through the nonsense-mediated decay pathway. It has been suggested that modulation by H3B-8800 is mediated by disruption of branchpoint sequence recognition by the SF3B complex as there is overall less preference for adenosine as the branchpoint nucleotide and a greater amount of sequences with weaker association to the SFB3 in introns retained with H3B-8800. It was found that 41 of 404 genes encoding spliceosome proteins contained GC-rich sequences whose retention was induced by H3B-8800. It is suggested that this is key to the specificity of H3B-8800's lethality as cells with spliceosome-mutant cells are dependent on the expression of wild-type spliceosome components for survival. Since cancer cells, as in myelodysplasia, experience SF3B1 mutations much more frequently than host cells, this allows H3B-8800 to be used to preferentially target these cells by inducing intron-retention in critical spliceosome component pre-mRNA leading to destruction of the now nonsense mature RNA ultimately cell-death due to the lack of these critical proteins.

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Pharmacodynamics
H3B-8800 preferentially targets cells with spliceosome complexes containing mutant splicing factor 3B1 (SF3B1) protein, modulating intron splicing leading to increased death in cancer cells while having little effect on the viability cells with wild-type SF3B1. Both normal and aberrant mature mRNA are suppressed in mutant and wild-type cells, the selectivity of the lethal effect is thought to be due to the presence of mutant SF3B1 and its implications rather than a change in mechanism or potency of effect on the mutant protein over the wild-type [A32749 . Since SF3B1 is frequently mutated in cancer, this allows preferential targeting of cancer cells over host cells.

C31H45N3O6

555.705508947372

555.33

C, 67.00; H, 8.16; N, 7.56; O, 17.27

1825302-42-8

1825302-42-8;

92135969

Yellow to orange solid

1.2±0.1 g/cm3

710.6±60.0 °C at 760 mmHg

383.6±32.9 °C

0.0±2.4 mmHg at 25°C

1.581

2.22

112Ų

2

8

6

40

928

6

O(C(N1CCN(C)CC1)=O)[C@H]1C=C[C@H](C)[C@@H](/C(=C/C=C/[C@@H](C)C2C=CC=CN=2)/C)OC(C[C@@H](CC[C@@]1(C)O)O)=O |t:12|

YOIQWBAHJZGRFW-WVRLKXNASA-N

InChI=1S/C31H45N3O6/c1-22(26-11-6-7-16-32-26)9-8-10-23(2)29-24(3)12-13-27(39-30(37)34-19-17-33(5)18-20-34)31(4,38)15-14-25(35)21-28(36)40-29/h6-13,16,22,24-25,27,29,35,38H,14-15,17-21H2,1-5H3/b9-8+,13-12+,23-10+/t22-,24+,25-,27+,29-,31-/m1/s1

(2S,3S,6S,7R,10R,E)-7,10-Dihydroxy-3,7-dimethyl-12-oxo-2-((R,2E,4E)-6-(pyridin-2-yl)hepta-2,4-dien-2-yl)oxacyclododec-4-en-6-yl 4-methylpiperazine-1-carboxylate

H3B-8800; H3B8800; RVT-2001; UNII-90YLS47BRX; RVT2001; 1825302-42-8; 90YLS47BRX; 1-Piperazinecarboxylic acid, 4-methyl-, (2S,3S,4E,6S,7R,10R)-7,10-dihydroxy-3,7-dimethyl-2-((1E,3E,5R)-1-methyl-5-(2-pyridinyl)-1,3-hexadien-1-yl)-12-oxooxacyclododec-4-en-6-yl ester; SCHEMBL17255784; EX-A8015; H3B 8800 [WHO-DD]; H3B 8800

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: ≥ 100 mg/mL (~180 mM)

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。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
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。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
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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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。