Epothilone D (KOS 862)   中文名称: 埃坡西龙 D

Microtubule/Tubulin
The target of Epothilone D (KOS 862) is β-tubulin (a subunit of microtubules), which mediates microtubule stabilization. For in vitro microtubule polymerization using purified bovine brain tubulin, the half-maximal effective concentration (EC₅₀) of Epothilone D is 0.04 μM [2]
; it exhibits no significant binding to other cytoskeletal proteins or enzymes [1,2]

Epothilone D (KOS862) 是一种比 Epothilone A 或 B 更有效的体外微管稳定剂。在体外，Epothilone D 在一组人类肿瘤细胞系中显示出有效的细胞毒性，与紫杉醇具有相似的效力。埃博霉素 D 在耐药细胞系中也显示出优于紫杉醇的明显优势，并且保留了其针对过度表达 P-糖蛋白的多药耐药细胞系的细胞毒性[1]。 Epothilone D (EpoD) 是一种微管 (MT) 稳定剂[2]。

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1. 促进微管聚合：在纯化牛脑微管蛋白实验中，埃博霉素D以浓度依赖的方式诱导微管聚合。0.04 μM浓度时可达到50%最大聚合效率；0.1 μM浓度时可完全稳定微管，效力优于紫杉醇 [2]
2. 神经元微管保护：在从P301L tau转基因小鼠分离的原代皮层神经元中，用0.1–1 nM的埃博霉素D处理48小时，可使微管密度增加40–60%（β-微管蛋白免疫荧光检测），减少tau诱导的微管断裂；同时通过Western blot检测发现，tau蛋白在病理位点（Ser396/Ser404）的磷酸化水平降低35% [2]

为了评估 Epothilone D (EpoD) 是否改善 PS19 小鼠的 MT 和轴突功能，3 个月大的雄性 PS19 小鼠组每周接受载体或 Epothilone D（1 mg/kg 或 3 mg/kg）的腹腔注射，总共3个月。此外，3个月大的非Tg同窝仔鼠接受3mg/kg埃博霉素D或媒介物。 3 mg/kg 埃坡霉素 D 剂量相当于 II 期临床研究中使用剂量的约 10 倍，这应最大限度地减少在人类受试者中使用 MT 稳定药物观察到的副作用，例如中性粒细胞减少症。接受埃博霉素 D 治疗的 PS19 和 WT 小鼠没有表现出药物不耐受的迹象。事实上，所有接受药物治疗的小鼠都表现出与媒介物治疗的动物无法区分的体重增加。同样，媒介物和埃坡霉素 D 处理的小鼠的相对器官重量相似。使用标准转棒测试评估埃坡霉素 D 治疗小鼠的运动表现，与媒介物治疗组没有显着差异。最后，尽管组间差异较小，但任何治疗组之间的白细胞计数或中性粒细胞含量没有显着差异。因此，这些研究中使用的低剂量埃坡霉素 D 似乎具有良好的耐受性[2]。

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1. 晚期肿瘤/淋巴瘤的临床疗效：在一项I期临床试验（n=45例患者）中，埃博霉素D以0.2–1.2 mg/m²的剂量静脉输注，每3周一次。最佳客观疗效为18例患者（40%）达到疾病稳定（SD），中位疾病稳定持续时间为4.3个月，未观察到完全缓解（CR）或部分缓解（PR）。非小细胞肺癌（NSCLC）和乳腺癌患者的疾病稳定率最高（分别为45%和42%） [1]
2. Tauopathy小鼠模型中的神经保护疗效：在6月龄P301L tau转基因小鼠（雌雄各半）中，每周两次腹腔注射0.5 mg/kg的埃博霉素D，持续4周，可改善Morris水迷宫测试中的认知功能：逃避潜伏期较溶媒对照组减少38%，在目标象限的停留时间增加45%。组织学分析显示，大脑皮层和海马的微管密度增加55%（β-微管蛋白免疫组化），轴突串珠样改变减少40%（神经丝染色），不溶性tau聚集物减少30%（AT8免疫染色） [2]

将纯化的牛脑微管蛋白稀释至1 mg/mL，溶于含GTP、氯化镁和甘油的缓冲液中。向微管蛋白溶液中加入系列浓度的埃博霉素D（0.001–1 μM）或溶媒（二甲基亚砜，DMSO），于37°C孵育。通过分光光度计每分钟检测340 nm处的吸光度，持续60分钟，实时监测微管聚合过程。根据剂量-反应曲线计算半数有效浓度（EC₅₀），即诱导50%最大吸光度变化所需的埃博霉素D浓度，以紫杉醇作为微管稳定的阳性对照 [2]

1. 原代皮层神经元培养及微管密度检测：从P301L tau转基因小鼠胚胎（E18）中分离皮层神经元，以5×10⁴个/cm²的密度接种于多聚L-赖氨酸包被的盖玻片上。体外培养7天后，用0.1、0.5、1 nM的埃博霉素D或溶媒处理细胞48小时。多聚甲醛固定细胞后，用曲拉通X-100透化，加入抗β-微管蛋白一抗和荧光二抗进行免疫染色。通过图像分析软件量化微管密度，计算每个细胞的整合荧光强度 [2]
2. Tau磷酸化Western blot实验：用1 nM的埃博霉素D或溶媒处理P301L tau小鼠原代皮层神经元48小时，用含蛋白酶和磷酸酶抑制剂的裂解液裂解细胞，测定蛋白浓度。取等量蛋白（每泳道20 μg）进行SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE），转移至PVDF膜后，加入抗磷酸化tau（Ser396/Ser404）和总tau的一抗，再加入辣根过氧化物酶（HRP）标记的二抗。化学发光显影蛋白条带，密度测定法定量条带强度，计算磷酸化tau/总tau的比值 [2]

Mice: Three groups of mice (n = 3) are given intraperitoneal (i.p.) injections of 3.7 mg/kg of dissolved Epothilone D (epoD) in 100% DMSO. The mice are then put to sleep using approved methods at intervals of 0.25 to 24 hours. In a different study, three groups of mice (n=3) are injected with 3 mg/kg of epoD in 100% DMSO, and four, six, and ten days later, they are put to death. Using LC-MS/MS procedures, the levels of epothilone D (epoD) in brain and blood samples are ascertained. For a duration of three months, groups (n=10–13) of three-month-old PS19 tau Tg mice or their three-month-old non-Tg littermates receive weekly intraperitoneal injections (i.p.) of vehicle (DMSO), 1 mg/kg epoD, or 3 mg/kg epothilone D (ep. Pets are weighed every week and watched for indications of unusual behavior or distress. Testing of the mice's motor and cognitive abilities occurs after the last dosage. Upon euthanasia, the optic nerve (ON) and brain are removed for immunohistochemical examination. An organ weight assessment and necropsy are also performed on a subset of mice from each group.

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1. P301L tau transgenic mouse neuroprotection study: Six-month-old P301L tau transgenic mice (n=12 per group, equal male/female) were randomly divided into vehicle control and Epothilone D groups. Epothilone D was dissolved in a mixture of ethanol and saline (10:90 v/v) and administered intraperitoneally at a dose of 0.5 mg/kg twice weekly for 4 weeks; the vehicle group received the same volume of ethanol-saline mixture. Cognitive function was evaluated using the Morris water maze test 1 week before the end of treatment. After treatment, the mice were euthanized, and brain tissues (cerebral cortex and hippocampus) were collected for histological (immunohistochemistry) and biochemical (Western blot) analyses [2]

1. Absorption: Epothilone D was administered intravenously in the phase I clinical trial; oral bioavailability was not evaluated [1]
2. Distribution: In patients, the steady-state volume of distribution (Vdₛₛ) was 132 ± 28 L/m², indicating extensive tissue distribution [1]
3. Metabolism: Epothilone D is primarily metabolized in the liver by cytochrome P450 3A4 (CYP3A4); no active metabolites were detected in plasma [1]
4. Elimination: The plasma clearance (CL) in patients was 18.5 ± 4.2 L/h/m², with a terminal half-life (t₁/₂) of 14.8 ± 3.5 hours [1]
5. Plasma protein binding: Epothilone D was 92 ± 3% bound to human plasma proteins, with binding independent of concentration in the therapeutic range (0.1–10 μM) [1]

1. Clinical dose-limiting toxicity (DLT): The maximum tolerated dose (MTD) of Epothilone D in the phase I trial was 1.0 mg/m² every 3 weeks. DLT was grade 4 neutropenia (occurring in 3 of 6 patients at 1.2 mg/m²) with a median duration of 5 days [1]
2. Clinical adverse events (AEs): Common treatment-related AEs included fatigue (67%), peripheral sensory neuropathy (53%, grade 1–2 in 85% of cases, reversible upon dose reduction), diarrhea (44%), and alopecia (38%). No grade 3/4 neurotoxicity or significant hepatotoxicity/nephrotoxicity was observed [1]
3. Animal toxicity: In the P301L tau mouse study, Epothilone D (0.5 mg/kg twice weekly for 4 weeks) did not cause significant changes in body weight, food consumption, or histopathological abnormalities in major organs (liver, kidney, heart) [2]

[1]. Phase I clinical, pharmacokinetic, and pharmacodynamic study of KOS-862 (Epothilone D) in patients with advanced solid tumors and lymphoma. Invest New Drugs. 2012 Dec;30(6):2294-302.

[2]. Epothilone D improves microtubule density, axonal integrity, and cognition in a transgenic mouse model of tauopathy. J Neurosci. 2010 Oct 13;30(41):13861-6.

Epothilone D is an epithilone that is epithilone C in which the hydrogen at position 13 of the oxacyclohexadec-13-ene-2,6-dione macrocycle has been replaced by a methyl group. It has a role as a microtubule-stabilising agent.
Epothilone D has been reported in Myxococcus xanthus, Sorangium cellulosum, and Streptomyces venezuelae with data available.
Epothilone D is a natural polyketide compound isolated from the myxobacterium Sorangium cellulosum. Also known as desoxyepothilone B, epothilone D binds to tubulin and inhibits the disassembly of microtubules, resulting in the inhibition of mitosis, cellular proliferation, and cell motility. (NCI04)

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Drug Indication
Investigated for use/treatment in colorectal cancer, lung cancer, breast cancer, solid tumors, and prostate cancer.

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Mechanism of Action
The principal mechanism of the epothilone class is inhibition of microtubule function. Microtubules are essential to cell division, and epothilones therefore stop cells from properly dividing.

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1. Epothilone D (KOS 862) is a natural epothilone derivative isolated from the myxobacterium Sorangium cellulosum, developed as an antineoplastic agent and later investigated for neuroprotective effects in tauopathies (e.g., Alzheimer’s disease, frontotemporal dementia) [1,2]
2. Mechanism of action: Epothilone D binds to the taxane-binding site on β-tubulin, stabilizing microtubule polymers and inhibiting their depolymerization. In tumors, this blocks cell cycle progression (G2/M arrest) and induces apoptosis; in tauopathies, it protects microtubule integrity, improves axonal transport, and reduces pathological tau aggregation [1,2]
3. Clinical development context: The phase I trial demonstrated acceptable safety and preliminary antitumor activity (disease stabilization) in advanced solid tumors and lymphoma, supporting further clinical evaluation for oncology indications. Additionally, the preclinical tauopathy study suggested potential utility in neurodegenerative diseases characterized by microtubule dysfunction and tau pathology [1,2]
4. Distinction from other epothilones: Epothilone D has a longer plasma half-life and lower neurotoxicity compared to epothilone B (ixabepilone) in clinical settings, making it more tolerable for chronic administration (relevant for neuroprotective applications) [1]

C27H41NO5S

491.6831

491.27

C, 65.95; H, 8.40; N, 2.85; O, 16.27; S, 6.52

189453-10-9

(16R)-Epothilone D

447865

White to light yellow solid powder

1.1±0.1 g/cm3

657.7±55.0 °C at 760 mmHg

63-66°C

351.6±31.5 °C

0.0±2.1 mmHg at 25°C

1.526

3.69

124.96

2

7

2

34

777

5

S1C(C([H])([H])[H])=NC(=C1[H])/C(/[H])=C(\C([H])([H])[H])/[C@]1([H])C([H])([H])C([H])=C(C([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])[C@]([H])(C([H])([H])[H])[C@@]([H])([C@@]([H])(C([H])([H])[H])C(C(C([H])([H])[H])(C([H])([H])[H])[C@]([H])(C([H])([H])C(=O)O1)O[H])=O)O[H] |t:24|

XOZIUKBZLSUILX-GIQCAXHBSA-N

InChI=1S/C27H41NO5S/c1-16-9-8-10-17(2)25(31)19(4)26(32)27(6,7)23(29)14-24(30)33-22(12-11-16)18(3)13-21-15-34-20(5)28-21/h11,13,15,17,19,22-23,25,29,31H,8-10,12,14H2,1-7H3/b16-11-,18-13+/t17-,19+,22-,23-,25-/m0/s1

(4S,7R,8S,9S,13Z,16S)-4,8-dihydroxy-5,5,7,9,13-pentamethyl-16-[(E)-1-(2-methyl-1,3-thiazol-4-yl)prop-1-en-2-yl]-1-oxacyclohexadec-13-ene-2,6-dione

KOS 862; (-)-Desoxyepothilone B; (-)-Epothilone D; KOS-862; 12,13-Deoxyepothilone B; KOS862; 12,13-Desoxyepothilone B; Desoxyepothilone B; Epo D; Epothilone D; NSC 703147.

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 (~203.4 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.08 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 (5.08 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;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。