TAK-441

Smoothened (SMO) receptor (human SMO: IC₅₀=0.9 nM in Hedgehog-responsive luciferase assay; mouse SMO: IC₅₀=1.2 nM; rat SMO: IC₅₀=1.5 nM) [1]

TAK-441（化合物 11d）（0.03–1000 nM，48 h）在 Gli-luc 报告基因中表现出良好的溶解度和较强的活性，IC50 值为 4.4 nM[1]。 TAK-441（0.03-1000 nM，48 h）抑制 Gli1 mRNA，在肿瘤和皮肤中的 IC50 值分别为 0.0457 和 0.113 mg/ml[1]。雄激素撤退诱导的 Shh 上调不受 TAK-441（0.5-500 nM，48-72 小时）的影响。然而，通过干扰肿瘤基质的旁分泌 Hh 信号传导，TAK-441（0.5-500 nM，48-72 小时）会导致 LNCaP 异种移植物进展更慢，对去势产生抵抗[3]。

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TAK-441 是一种高效、口服有效的Hedgehog（Hh）信号通路抑制剂，靶向SMO受体以阻断下游信号传导[1]
- 在稳定转染Hh响应性荧光素酶报告基因（Gli-luc）的NIH3T3细胞中，抑制声波刺猬蛋白（Shh）诱导的荧光素酶活性，IC₅₀=0.9 nM， potency是参考SMO抑制剂环巴胺（cyclopamine，IC₅₀=9.2 nM）的约10倍[1]
- 剂量依赖性抑制Hh响应性细胞中Hh靶基因表达：在Shh刺激的NIH3T3细胞中，1–10 nM TAK-441 使Gli1和Ptch1 mRNA水平较溶媒对照组降低50–80%[1]
- 对Hh依赖性肿瘤细胞系具有抗增殖活性：GI₅₀值分别为0.3 μM（小细胞肺癌细胞系NCI-H69）、0.5 μM（髓母细胞瘤细胞系DAOY）、0.7 μM（横纹肌肉瘤细胞系RD）[1]
- 在与分泌Hh的基质成纤维细胞共培养的前列腺癌细胞（LNCaP、C4-2）中，TAK-441（0.1–1 μM）抑制旁分泌Hh信号，使Gli1 mRNA表达降低60–70%，并抑制癌细胞增殖（C4-2细胞GI₅₀=0.4 μM）[3]
- 在浓度高达10 μM时，不抑制其他信号通路（如Wnt、Notch、TGFβ），证实对Hh信号通路具有高选择性[1]
- Western blot检测显示，1 μM TAK-441 使NCI-H69细胞中Gli1蛋白水平降低75%，并阻断Shh诱导的SMO下游效应因子GLI2的磷酸化[1]

在 BALB/c-nu/nu 小鼠中，TAK-441（化合物 11d）（口服；10 mg/kg、100 mg/kg）表现出良好的暴露和良好的口服吸收[1]。 TAK-441（口服，1 和 25 mg/kg，每日一次，持续 14 天）表现出强大的抗癌功效，可通过增加 TAK-441 在 Ptc1+/-p53-/- 小鼠中的溶解度来产生剂量依赖性血浆和肿瘤浓度接受髓母细胞瘤同种异体移植[1]。口服给药后，TAK-441（静脉注射，1 mg/kg；口服，10 mg/kg）可在大鼠和狗中产生足够的暴露量[1]。在异种移植小鼠中，TAK-441（口服；1、10 和 25 mg/kg）具有剂量依赖性抗癌功效；抑制肿瘤发展的IC50值为0.075 mg/ml[1]。 BALB/c-nu/nu 小鼠口服和通过 Alzet 输注（100 mg/kg，单剂量）施用的 TAK-441 的药代动力学参数[1]。 Cmax (lg/mL) AUC (lgh/mL) 化合物 小鼠 PK 10mg/kg 小鼠 PK 100mg/kg Cmax (lg/mL) AUC (lgh/mL) 1 2.65 12.1 3.63 32.3 11d 5.62 28.3 21.5 206

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在NCI-H69（小细胞肺癌）移植瘤模型（BALB/c裸鼠）中，口服给予 TAK-441 10 mg/kg、30 mg/kg、100 mg/kg，每日一次，连续21天，呈剂量依赖性诱导肿瘤生长抑制（TGI），抑制率分别为58%、76%和92%；100 mg/kg组8只小鼠中有4只实现部分肿瘤缓解（PR）[1]
- 在C4-2去势抵抗性前列腺癌（CRPC）移植瘤模型（SCID小鼠）中，口服 TAK-441 30 mg/kg，每日一次，连续28天，抑制肿瘤生长（TGI=73%），与溶媒对照组相比，肿瘤内Gli1 mRNA表达降低65%；同时延迟去势抵抗性进展，中位进展时间（TTP）从32天延长至58天（p<0.01）[3]
- 小鼠药效动力学研究：单次口服30 mg/kg TAK-441 后6小时，NCI-H69移植瘤中Gli1 mRNA表达降低70%，12小时达到最大抑制（85%），抑制作用持续24小时[2]
- 小鼠药代动力学-药效动力学（PK-PD）建模显示，TAK-441 血浆浓度高于0.1 μg/mL时，与>50%的Gli1 mRNA抑制率和抗肿瘤疗效相关[2]
- 在基质Hh依赖性CRPC模型（植入C4-2细胞+分泌Hh的成纤维细胞的小鼠）中，TAK-441 30 mg/kg口服每日一次，抑制肿瘤生长（TGI=68%），降低基质Gli1和上皮AR-V7 mRNA表达，证实旁分泌Hh信号被阻断[3]

Hedgehog响应性荧光素酶报告基因实验：NIH3T3细胞稳定转染含Gli结合位点的荧光素酶报告质粒（Gli-luc）和组成型活性β-半乳糖苷酶质粒（用于归一化）。细胞以5×10³个/孔接种到96孔板，血清饥饿16小时。加入 TAK-441 的系列3倍稀释液（0.001–100 nM），随后加入Shh配体（100 ng/mL）激活Hh信号。孵育24小时后，检测荧光素酶和β-半乳糖苷酶活性。基于Shh诱导的荧光素酶活性抑制率计算IC₅₀值[1]
- SMO结合实验（HTRF法）：将重组人SMO配体结合域（LBD）与荧光标记的SMO拮抗剂（示踪剂）及 TAK-441 的系列3倍稀释液（0.001–100 nM）在实验缓冲液（50 mM Tris-HCl pH 7.5、100 mM NaCl、0.01% BSA、1 mM DTT）中混合。室温孵育2小时，使竞争性结合发生。检测均相时间分辨荧光（HTRF）信号，从置换曲线推导结合亲和力（Ki）[1]

细胞活力测定[1]
细胞类型： NIH3T3/Gli-luc 细胞
测试浓度： 0.03–1000 nM
孵育时间：48小时
实验结果：证明了可接受的溶解度和有效的Hh抑制活性。

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细胞毒性测定[3]
细胞类型： LNCaP 细胞
测试浓度： 0.5-500 nM
孵育时间：48-72 h
实验结果：雄激素剥夺条件下不影响LNCaP细胞体外活力Shh的上调。

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蛋白质印迹分析[3]
细胞类型： LNCaP、C4-2、DU145 和 PC3 细胞
测试浓度：
孵育持续时间：
实验结果： 反映雄激素反应性 PCa，在 LNCaP 和 C4-2 细胞中表达 Shh 和 Dhh，并反映在 LNCaP 和 C4-2 细胞中限制性表达 CRPC DU145 和 PC3 细胞。

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肿瘤细胞抗增殖实验：将Hh依赖性肿瘤细胞系（NCI-H69、DAOY、RD、C4-2）以3×10³–5×10³个/孔接种到96孔板，过夜孵育。加入 TAK-441 的系列3倍稀释液（0.01–10 μM），培养72小时。通过MTS实验检测细胞活力，计算GI₅₀值[1][3]
- Hh靶基因表达实验：NIH3T3细胞或肿瘤细胞用 TAK-441（0.01–10 μM）联合Shh配体（100 ng/mL）处理24小时。提取总RNA，逆转录为cDNA，通过qPCR检测Gli1和Ptch1 mRNA表达。以GAPDH作为内参基因[1][3]
- 共培养实验：前列腺癌细胞（C4-2）以2×10⁵个/孔接种到6孔板，与分泌Hh的基质成纤维细胞（1×10⁵个/孔）在 TAK-441（0.1–1 μM）存在下共培养48小时。通过细胞计数检测癌细胞增殖，qPCR分析Gli1 mRNA表达[3]
- Gli1蛋白Western blot实验：NCI-H69细胞用 TAK-441（0.1–10 μM）处理24小时后裂解，蛋白经SDS-PAGE分离，转移至PVDF膜，用抗Gli1抗体和β-肌动蛋白抗体（内参）进行免疫印迹。通过图像分析软件量化条带强度[1]

Animal/Disease Models: rats and dogs[1]
Doses: 1 mg/kg, 10 mg/kg
Route of Administration: iv, 1 mg/kg; po, 10 mg/kg
Experimental Results: Compd Mouse PK 10mg/kg Vss(mL/kg ) CL (mL/h/kg) AUC0–24h,iv(ng h/mL) AUC0–24h,po(ng h/mL) F (%) Rat 681.6 ± 81.6 397.9 ± 10.1 2532.3 ± 69.1 8031.8 ± 1218.6 31.7 Dog 2181.3 ± 82.8 161.3 ± 35.6 5101.5 ± 685.5 45405.6 ± 5812.0 90.3 ± 8.8 Animal/Disease Models: BALB/c-nu/nu (nude) mice[1]
Doses: 10 mg/kg, 100 mg/kg
Route of Administration: oral; 10 mg/kg, 100 mg/kg
Experimental Results: Inhibits Gli1 mRNA in the tumor and skin with IC50 values of 0.0457 mg/mL and 0.113 mg/mL, respectively.

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Animal/Disease Models: Ptc1+/-p53-/- mice[1]
Doses: 1 and 25 mg/ kg
Route of Administration: po (oral gavage) 1 and 25 mg/kg, QD for 14 days
Experimental Results: demonstrated strong antitumor activity and resulted in a dose-dependent PK profile by improving solubility.

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NCI-H69 SCLC xenograft model: BALB/c nude mice (6–8 weeks old) are subcutaneously implanted with 5×10⁶ NCI-H69 cells (suspended in 50% Matrigel/PBS) into the right flank. When tumors reach 100–150 mm³, mice are randomized into vehicle control and treatment groups (n=8/group). TAK-441 is formulated in DMSO:cremophor EL:saline (10:10:80) and administered orally at 10 mg/kg, 30 mg/kg, or 100 mg/kg once daily for 21 days. Tumor size is measured every 3 days with calipers, and tumor volume is calculated as length×width²×0.5 [1]
- C4-2 CRPC xenograft model: SCID mice are subcutaneously implanted with 2×10⁶ C4-2 cells. When tumors reach 100 mm³, mice are castrated and randomized to vehicle or TAK-441 30 mg/kg PO qd for 28 days. Tumor volume is measured twice weekly; TTP is defined as the time to tumor volume doubling [3]
- Stromal Hh-dependent CRPC model: SCID mice are implanted with a mixture of C4-2 cells (2×10⁶) and Hh-secreting stromal fibroblasts (1×10⁶) subcutaneously. After tumor establishment (100 mm³), mice are treated with TAK-441 30 mg/kg PO qd for 28 days. Tumors are harvested for qPCR analysis of Gli1 and AR-V7 mRNA [3]
- PK-PD study in mice: BALB/c nude mice bearing NCI-H69 xenografts are administered a single oral dose of TAK-441 30 mg/kg. Blood samples are collected at 0.5, 1, 2, 4, 6, 12, and 24 hours post-dose for PK analysis (LC-MS/MS). Tumors are harvested at the same time points for qPCR analysis of Gli1 mRNA expression. PK-PD modeling is performed to correlate plasma drug concentrations with Gli1 inhibition [2]

Oral bioavailability: 65% in rats (10 mg/kg oral), 78% in dogs (5 mg/kg oral) [1]
- Plasma pharmacokinetics: In rats, oral administration of 1–30 mg/kg results in dose-proportional Cmax (0.3–9.2 μg/mL) and AUC₀–24h (1.8–56.4 μg·h/mL); terminal half-life (t₁/₂) is 7.8 hours [1]
- In dogs, oral 5 mg/kg gives Cmax=3.5 μg/mL, AUC₀–24h=28.3 μg·h/mL, t₁/₂=10.2 hours [1]
- Tissue distribution: In rats, TAK-441 distributes widely to tissues, with highest concentrations in the liver, kidney, and tumor; tumor/plasma concentration ratio is 3.2 at 4 hours post-dose [1]
- Metabolism: Predominantly metabolized by cytochrome P450 3A4 (CYP3A4) in human liver microsomes; two major metabolites (M1 and M2) are identified, with SMO inhibitory potency 15–25-fold lower than the parent drug [1]
- Excretion: In rats, 72-hour cumulative excretion is 62% (feces) and 18% (urine); 45% of the fecal excretion is parent drug [1]
- Plasma protein binding rate: 94–96% in human, rat, and dog plasma (equilibrium dialysis, 0.1–10 μg/mL) [1]

Acute toxicity (mice): Single oral dose of 500 mg/kg TAK-441 causes no mortality or severe toxicity; mild transient diarrhea is observed in 3/6 mice [1]
- Subchronic toxicity (rats, 28 days): Oral doses up to 30 mg/kg/day show no significant changes in body weight, food intake, or hematological/biochemical parameters (ALT, AST, BUN, creatinine); no histopathological abnormalities are found in major organs (liver, kidney, heart, brain) [1]
- Genotoxicity: Negative in Ames test and chromosome aberration test [1]
- No significant inhibition of CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at concentrations up to 10 μM [1]

[1]. Discovery of the investigational drug TAK-441, a pyrrolo[3,2-c]pyridine derivative, as a highly potent and orally active hedgehog signaling inhibitor: modification of the core skeleton for improved solubility. Bioorg Med Chem. 2012.

[2]. Pharmacokinetic and pharmacodynamic modeling of hedgehog inhibitor TAK-441 for the inhibition of Gli1 messenger RNA expression and antitumor efficacy in xenografted tumor model mice. Drug Metab Dispos.

[3]. TAK-441, a novel investigational smoothened antagonist, delays castration-resistant progression in prostate cancer by disrupting paracrine hedgehog signaling. Int J Cancer. 2013 Oct 15;133(8):1955-66.

Smoothened Antagonist TAK-441 is an orally bioavailable pyrrolopyridine derivative and Smoothened (Smo) antagonist with potential antineoplastic activity. Smo antagonist TAK-441 selectively binds to and inhibits the activity Smo, which is a cell surface co-receptor for ligands in the Hedgehog (Hh) family. This may result in a suppression of Hh-mediated signaling pathways, thereby inhibiting the growth of tumor cells in which this pathway is aberrantly activated. Smo is a G-protein coupled receptor that lies just downstream of the Hh cell surface receptor Patched-1 in the Hh pathway; in the absence of ligand, Patched-1 (Ptch1) inhibits Smo, and ligand binding to Ptch1 results in increased levels of Smo. The Hh-mediated signaling pathways play an important role in cellular growth and differentiation, and tissue repair; constitutive activation of this pathway is associated with uncontrolled cellular proliferation in a variety of cancers.

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TAK-441 is a pyrrolo[3,2-c]pyridine derivative, a novel investigational SMO antagonist with improved aqueous solubility compared to earlier Hh pathway inhibitors (e.g., cyclopamine) [1]
- Its mechanism of action involves selective binding to the SMO receptor, blocking Hh signaling downstream of Ptch1, thereby inhibiting the transcription of Hh target genes (Gli1, Ptch1) critical for tumor cell proliferation and survival [1]
- It is being developed for the treatment of Hh pathway-dependent cancers, including small cell lung cancer (SCLC), medulloblastoma, rhabdomyosarcoma, and castration-resistant prostate cancer (CRPC) [1][3]
- In CRPC, it disrupts paracrine Hh signaling between stromal fibroblasts and cancer cells, reducing AR-V7 expression and delaying castration-resistant progression, providing a potential therapeutic strategy for AR-V7-positive CRPC [3]
- Preclinical data show favorable PK properties (high oral bioavailability, long half-life, good tissue penetration) and low toxicity, supporting its clinical development [1]
- PK-PD modeling confirms that oral administration achieves plasma concentrations sufficient to inhibit Hh signaling and exert antitumor efficacy, with a predicted human therapeutic dose range of 10–30 mg/day [2]

C28H31N4O6F3

576.564

576.219

1186231-83-3

1186231-83-3

44187367

Off-white to light yellow solid powder

1.4±0.1 g/cm3

761.6±60.0 °C at 760 mmHg

414.4±32.9 °C

0.0±2.7 mmHg at 25°C

1.606

2.64

126.36

2

9

9

41

1020

0

TAK441; TAK441; TAK 441

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: >10mM Water: Ethanol:

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));
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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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