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 (compound 11d) (0.03–1000 nM, 48 h) exhibits good solubility and strong activity in the Gli-luc reporter, with an IC50 value of 4.4 nM[1]. With IC50 values of 0.0457 and 0.113 mg/ml in the tumor and skin, respectively, TAK-441 (0.03–1000 nM, 48 h) inhibits Gli1 mRNA[1]. The androgen withdrawal-induced Shh up-regulation is not affected by TAK-441 (0.5-500 nM, 48-72 h). However, by interfering with paracrine Hh signaling with the tumor stroma, TAK-441 (0.5-500 nM, 48-72 h) causes LNCaP xenografts to progress more slowly, resistant to castration[3].

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TAK-441 is a highly potent, orally active inhibitor of the Hedgehog (Hh) signaling pathway, targeting the SMO receptor to block downstream signaling [1]
- In NIH3T3 cells stably transfected with a Hh-responsive luciferase reporter gene (Gli-luc), it inhibits Sonic Hedgehog (Shh)-induced luciferase activity with an IC₅₀ of 0.9 nM, showing ~10-fold higher potency than the reference SMO inhibitor cyclopamine (IC₅₀=9.2 nM) [1]
- It dose-dependently inhibits Hh target gene expression in Hh-responsive cells: in Shh-stimulated NIH3T3 cells, 1–10 nM TAK-441 reduces Gli1 and Ptch1 mRNA levels by 50–80% compared to vehicle control [1]
- Exhibits antiproliferative activity against Hh-dependent cancer cell lines: GI₅₀ values are 0.3 μM (SCLC cell line NCI-H69), 0.5 μM (medulloblastoma cell line DAOY), and 0.7 μM (rhabdomyosarcoma cell line RD) [1]
- In prostate cancer cells (LNCaP, C4-2) co-cultured with Hh-secreting stromal fibroblasts, TAK-441 (0.1–1 μM) inhibits paracrine Hh signaling, reducing Gli1 mRNA expression by 60–70% and suppressing cancer cell proliferation (GI₅₀=0.4 μM for C4-2 cells) [3]
- It does not inhibit other signaling pathways (e.g., Wnt, Notch, TGFβ) at concentrations up to 10 μM, confirming high selectivity for Hh signaling [1]
- Western blot analysis shows that 1 μM TAK-441 reduces Gli1 protein levels in NCI-H69 cells by 75% and blocks Shh-induced phosphorylation of SMO downstream effector GLI2 [1]

In BALB/c-nu/nu mice, TAK-441 (compound 11d) (oral; 10 mg/kg, 100 mg/kg) exhibits a favorable exposure and good oral absorption[1]. Strong anticancer efficacy is exhibited by TAK-441 (oral, 1 and 25 mg /kg, QD for 14 days), which can produce dose-dependent plasma and tumor concentrations by increasing TAK-441's solubility in Ptc1+/-p53-/-mice receiving medulloblastoma allografts[1]. After oral dosing, TAK-441 (iv, 1 mg/kg; po, 10 mg/kg) can produce enough exposure in rats and dogs[1]. In xenografted mice, TAK-441 (oral; 1, 10, and 25 mg/kg) has dose-dependent anticancer efficacy; the IC50 value for the suppression of tumor development is 0.075 mg/ml[1]. Pharmacokinetic parameters of TAK-441 administered orally and via Alzet infusion (100 mg/kg, single dose) in BALB/c-nu/nu mice[1]. Cmax (lg/mL) AUC (lgh/mL) Compd Mouse PK 10mg/kg Mouse 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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In NCI-H69 (SCLC) xenograft model (BALB/c nude mice): Oral administration of TAK-441 at 10 mg/kg, 30 mg/kg, and 100 mg/kg once daily for 21 days results in dose-dependent tumor growth inhibition (TGI) of 58%, 76%, and 92%, respectively; 100 mg/kg group achieves partial tumor regression (PR) in 4/8 mice [1]
- In C4-2 castration-resistant prostate cancer (CRPC) xenograft model (SCID mice): Oral TAK-441 30 mg/kg once daily for 28 days inhibits tumor growth (TGI=73%) and reduces intratumoral Gli1 mRNA expression by 65% compared to vehicle; it also delays castration-resistant progression, with median time to progression (TTP) increased from 32 days to 58 days (p<0.01) [3]
- In pharmacodynamic studies (mice): Single oral dose of 30 mg/kg TAK-441 reduces Gli1 mRNA expression in NCI-H69 xenografts by 70% at 6 hours post-dose, with maximal inhibition (85%) at 12 hours and sustained suppression for 24 hours [2]
- Pharmacokinetic-pharmacodynamic (PK-PD) modeling in mice shows that plasma concentrations of TAK-441 above 0.1 μg/mL correlate with >50% inhibition of Gli1 mRNA expression and antitumor efficacy [2]
- In stromal Hh-dependent CRPC model (mice implanted with C4-2 cells + Hh-secreting fibroblasts), TAK-441 30 mg/kg PO qd inhibits tumor growth (TGI=68%) and reduces stromal Gli1 and epithelial AR-V7 mRNA expression, confirming disruption of paracrine Hh signaling [3]

Hedgehog-responsive luciferase reporter assay: NIH3T3 cells are stably transfected with a luciferase reporter plasmid containing Gli-binding sites (Gli-luc) and a constitutively active β-galactosidase plasmid (for normalization). Cells are seeded in 96-well plates (5×10³ cells/well) and serum-starved for 16 hours. Serial 3-fold dilutions of TAK-441 (0.001–100 nM) are added, followed by Shh ligand (100 ng/mL) to activate Hh signaling. After 24 hours of incubation, luciferase and β-galactosidase activities are measured. IC₅₀ value is calculated based on the inhibition of Shh-induced luciferase activity [1]
- SMO binding assay (HTRF-based): Recombinant human SMO ligand-binding domain (LBD) is mixed with a fluorescently labeled SMO antagonist (tracer) and serial 3-fold dilutions of TAK-441 (0.001–100 nM) in assay buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.01% BSA, 1 mM DTT). The mixture is incubated at room temperature for 2 hours to allow competitive binding. Homogeneous time-resolved fluorescence (HTRF) signal is measured, and binding affinity (Ki) is derived from displacement curves [1]

Cell Viability Assay[1]
Cell Types: NIH3T3/Gli-luc cells
Tested Concentrations: 0.03–1000 nM
Incubation Duration: 48 h
Experimental Results: demonstrated acceptable solubility and potent Hh inhibitory activity.

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Cell Cytotoxicity Assay[3]
Cell Types: LNCaP cells
Tested Concentrations: 0.5-500 nM
Incubation Duration: 48-72 h
Experimental Results: Did not affect up-regulation of Shh of in vitro viability of LNCaP cells under androgen-deprivedconditionsin.

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Western Blot Analysis[3]
Cell Types: LNCaP, C4-2, DU145 and PC3 cells
Tested Concentrations:
Incubation Duration:
Experimental Results: Reflected androgen-responsive PCa and express both Shh and Dhh in LNCaP and C4-2 cells and reflect restricted Shh expression of CRPC in DU145 and PC3 cells.

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Cancer cell antiproliferation assay: Hh-dependent cancer cell lines (NCI-H69, DAOY, RD, C4-2) are seeded in 96-well plates (3×10³–5×10³ cells/well) and incubated overnight. Serial 3-fold dilutions of TAK-441 (0.01–10 μM) are added, and cells are cultured for 72 hours. Cell viability is detected by MTS assay, and GI₅₀ values are calculated [1][3]
- Hh target gene expression assay: NIH3T3 cells or cancer cells are treated with TAK-441 (0.01–10 μM) plus Shh ligand (100 ng/mL) for 24 hours. Total RNA is extracted, reverse-transcribed into cDNA, and qPCR is performed to detect Gli1 and Ptch1 mRNA expression. GAPDH is used as an internal reference [1][3]
- Co-culture assay: Prostate cancer cells (C4-2) are seeded in 6-well plates (2×10⁵ cells/well) and co-cultured with Hh-secreting stromal fibroblasts (1×10⁵ cells/well) in the presence of TAK-441 (0.1–1 μM) for 48 hours. Cancer cell proliferation is measured by cell counting, and Gli1 mRNA expression is analyzed by qPCR [3]
- Western blot for Gli1 protein: NCI-H69 cells are treated with TAK-441 (0.1–10 μM) for 24 hours. Cells are lysed, proteins are separated by SDS-PAGE, transferred to PVDF membranes, and probed with anti-Gli1 antibody and β-actin antibody (loading control). Band intensity is quantified using image analysis software [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 orally) and 78% in dogs (5 mg/kg orally) [1] - Plasma pharmacokinetics: In rats, after oral administration of 1–30 mg/kg, Cmax showed dose-proportional variation (0.3–9.2 μg/mL), and AUC₀–24h showed dose-proportional variation (1.8–56.4 μg·h/mL); the terminal half-life (t₁/₂) was 7.8 hours [1] - In dogs, after oral administration of 5 mg/kg, Cmax = 3.5 μg/mL, AUC₀–24h = 28.3 μg·h/mL, and t₁/₂ = 10.2 hours [1] - Tissue distribution: In rats, TAK-441 It is widely distributed in various tissues, with the highest concentrations in the liver, kidneys and tumors; the tumor/plasma concentration ratio was 3.2 4 hours after administration [1]
- Metabolism: It is mainly metabolized in human liver microsomes by cytochrome P450 3A4 (CYP3A4); two major metabolites (M1 and M2) have been identified, with SMO inhibitory efficacy 15-25 times lower than that of the parent drug [1]
- Excretion: In rats, the cumulative excretion rate was 62% (feces) and 18% (urine) after 72 hours; 45% of the fecal excretion was the parent drug [1]
- Plasma protein binding: 94-96% in human, rat and canine plasma (equilibrium dialysis, 0.1-10 μg/mL) [1]

Acute toxicity (mice): A single oral dose of 500 mg/kg TAK-441 did not cause death or serious toxicity; 3 out of 6 mice experienced mild, transient diarrhea [1] - Subchronic toxicity (rats, 28 days): Oral doses up to 30 mg/kg/day did not significantly change body weight, food intake, or hematological/biochemical parameters (ALT, AST, BUN, creatinine); no histopathological abnormalities were found in major organs (liver, kidney, heart, brain) [1] - Genotoxicity: Ames test and chromosome aberration test results were negative [1] - No significant inhibition of CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) was observed 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.

TAK-441, a highly bioavailable orally bioavailable pyrrolopyridine derivative, is a smoothed (Smo) antagonist with potential antitumor activity. TAK-441 selectively binds to and inhibits the activity of Smo, a cell surface co-receptor for Hedgehog (Hh) family ligands. This may lead to suppression of Hh-mediated signaling pathways, thereby inhibiting the growth of tumor cells with aberrant activation of these pathways. Smo is a G protein-coupled receptor located downstream of the Hh cell surface receptor Patched-1 in the Hh pathway; in the absence of a ligand, Patched-1 (Ptch1) inhibits Smo, while ligand binding to Ptch1 leads to elevated Smo levels. Hh-mediated signaling pathways play a crucial role in cell growth, differentiation, and tissue repair. Constitutive activation of this pathway is associated with uncontrolled cell proliferation in various cancers.

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TAK-441 is a pyrrolo[3,2-c]pyridine derivative and a novel investigational SMO antagonist with improved water solubility compared to earlier Hh pathway inhibitors such as cyclopamine [1]
- Its mechanism of action involves selectively binding to the SMO receptor, blocking Hh signaling downstream of Ptch1, thereby inhibiting the transcription of Hh target genes (Gli1, Ptch1) that are crucial 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, reduces AR-V7 expression, and delays castration-resistant progression, providing a potential therapeutic strategy for AR-V7-positive CRPC [3]
- Preclinical data showed that it had good pharmacokinetic properties (high oral bioavailability, long half-life, good tissue penetration) and low toxicity, supporting its clinical development [1]. Pharmacokinetic-pharmacodynamic models confirmed that oral administration could achieve plasma concentrations sufficient to inhibit the Hh signaling pathway and exert antitumor effects, 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

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:

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)