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Purity: ≥98%
TAK-960 (TAK960) is a novel, investigational, orally bioavailable, and selective inhibitor of polo-like kinase 1 (PLK1) with anticancer activity. TAK-960 also exhibits inhibitory activities against PLK2 and PLK3, with IC50s of 16.9 and 50.2 nM, respectively. It inhibits PLK1 with an IC50 of 0.8 nM at 10 μM ATP.Serine/threonine protein kinase PLK1 is essential for several processes in mitosis. Human PLK1 is a promising target for anticancer therapy because it has been demonstrated to be overexpressed in a variety of human cancers and because elevated levels of PLK1 have been linked to a poor prognosis. Multiple tumor cell lines, including those that express multidrug-resistant protein 1 (MDR1), have demonstrated TAK-960's efficacy. TAK-960 treatment resulted in G(2)-M cell accumulation, abnormal polo mitosis morphology, and elevated phosphorylation of histone H3 (pHH3) in vitro and in vivo, all of which were consistent with PLK1 inhibition. TAK-960 did not affect nondividing normal cells (EC(50) >1,000 nmol/L), but it did inhibit the proliferation of several cancer cell lines, with mean EC(50) values ranging from 8.4 to 46.9 nmol/L. In the tested cell lines, there was no correlation found between the potency of TAK-960 and the mutation status of TP53, KRAS, and MDR1 expression. Oral administration of TAK-960 in animal models resulted in a dose-dependent increase in pHH3 and a significant inhibition of the growth of HT-29 colorectal cancer xenografts. The administration of TAK-960 once daily demonstrated noteworthy effectiveness against various tumor xenograft models, such as a disseminated leukemia model and an adriamycin/paclitaxel-resistant xenograft model. TAK-960 is currently being evaluated clinically in patients with metastatic cancer.
| Targets |
PLK1 (IC50 = 0.8 nM); PLK2 (IC50 = 16.9 nM); PLK3 (IC50 = 50.2 nM); FAK/PTK2 (IC50 = 19.6 nM); MLCK/MYLK (IC50 = 25.6 nM); FES/FPS (IC50 = 58.2 nM)
TAK-960 exhibited activity in a number of tumor cell lines, including those that express MDR1, the multidrug resistance protein. (Source: ) In line with PLK1 inhibition, TAK-960 treatment results in an increase in phosphorylation of histone H3 (pHH3), an accumulation of G2/M cells, and aberrant "polo" mitosis morphology. (Source: ) TAK-960 does not affect non-dividing normal cells (EC50 >1,000 nM), but it does inhibit the proliferation of several cancer cell lines, with mean EC50 values ranging from 8.4 to 46.9 nM. In the tested cell lines, there is no correlation between the potency of TAK-960 and the mutation status of TP53, KRAS, or MDR1 expression.[1] |
|---|---|
| ln Vitro |
TAK-960 exhibited activity in a number of tumor cell lines, including those that express MDR1, the multidrug resistance protein. (Source: ) In line with PLK1 inhibition, TAK-960 treatment results in an increase in phosphorylation of histone H3 (pHH3), an accumulation of G2/M cells, and aberrant "polo" mitosis morphology. (Source: ) TAK-960 does not affect non-dividing normal cells (EC50 >1,000 nM), but it does inhibit the proliferation of several cancer cell lines, with mean EC50 values ranging from 8.4 to 46.9 nM. In the tested cell lines, there is no correlation between the potency of TAK-960 and the mutation status of TP53, KRAS, or MDR1 expression.[1]
TAK-960 inhibited the proliferation of 18 human cancer cell lines in a concentration-dependent manner, with mean EC50 values ranging from 8.4 to 46.9 nmol/L. The antiproliferative activity was independent of TP53 or KRAS mutation status and multidrug-resistant protein 1 (MDR1) expression. [1] TAK-960 treatment of HT-29 colorectal cancer cells caused concentration-dependent accumulation of cells in G2-M phase, as shown by flow cytometry DNA analysis. [1] In HT-29 cells, TAK-960 treatment increased phosphorylation of histone H3 (pHH3) at serine 10, a mitotic marker, with an EC50 of 9.8 nmol/L at 24 hours, which correlated with the loss of cell viability (EC50 = 8.4 nmol/L at 72 hours). [1] Immunofluorescence microscopy in HT-29 cells revealed that TAK-960 treatment induced aberrant mitotic spindle morphology, including monopolar spindles characteristic of polo spindle morphology, at concentrations of 100 nmol/L or higher. [1] TAK-960 reduced the viability of proliferating normal human lung fibroblast cells (MRC5), but viability remained above 50% at 1000 nmol/L. It did not affect the viability of quiescent MRC5 cells (EC50 >1000 nmol/L). [1] TAK-960 inhibited proliferation of the doxorubicin/paclitaxel-resistant, MDR1-overexpressing leukemia cell line K562ADR with similar potency (EC50) as its parent K562 cell line, whereas doxorubicin and paclitaxel showed significantly reduced activity against K562ADR. [1] |
| ln Vivo |
Oral TAK-960 administration in animal models causes a dose-dependent increase in pHH3 and a significant inhibition of the growth of HT-29 colorectal cancer xenografts.[1] Once-daily TAK-960 treatment shows notable efficacy against a variety of tumor xenografts, such as a disseminated leukemia model and a xenograft model resistant to doxorubicin and paritamol.[1]
Oral administration of TAK-960 significantly inhibited tumor growth in subcutaneous xenograft models of HT-29 colorectal cancer using various regimens (e.g., 10 mg/kg once daily for 21 days induced complete regression in 3 of 5 mice). [1] TAK-960 (10 mg/kg orally once daily for 14 days) showed substantial antitumor activity and was well-tolerated in multiple solid tumor xenograft models, including HCT116 (colorectal), PC-3 (prostate), BT474 (breast), A549 (lung), NCI-H1299 (lung), NCI-H1975 (lung), and A2780 (ovary) cancers. [1] In a subcutaneous MV4-11 leukemia xenograft model, TAK-960 (10 mg/kg orally once daily for 14 days) significantly inhibited tumor growth. [1] In a disseminated leukemia model using MV4-11luc cells, TAK-960 (7.5 mg/kg orally once daily for 9 days) significantly increased median survival compared to vehicle (39.5 vs. 25 days) and inhibited tumor growth as measured by in vivo imaging. [1] In the MDR1-overexpressing K562ADR leukemia xenograft model, TAK-960 (10 mg/kg orally once daily, 6 days/week for 2 weeks) inhibited tumor growth, whereas paclitaxel (10 mg/kg intraperitoneally, 5 days/week for 2 weeks) did not. [1] A single oral dose of TAK-960 (30 mg/kg) increased tumor pHH3 levels (a PD marker of mitotic arrest) in both K562 and MDR1-expressing K562ADR xenografts, whereas paclitaxel induced pHH3 only in K562 but not in K562ADR tumors. [1] |
| Enzyme Assay |
The TR-FRET assay, which measures the ATP-dependent phosphorylation of a biotinylated substrate peptide corresponding to residues 2470 through 2488 of the mammalian target of rapamycin protein (Biotin-AGAGTVPESIHSFIGDGLV), is used to evaluate the inhibitory activity of TAK-960. Using HotSpotSM technology, 288 kinases are screened for TAK-960 inhibition (1 μM), and the IC50 values of the kinases that pass the test are found.
The inhibitory activity of TAK-960 against PLK1 was assessed using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. This assay measures the ATP-dependent phosphorylation of a biotinylated substrate peptide corresponding to a segment of the mTOR protein. The kinase domain of PLK1 was incubated with TAK-960 at varying concentrations in the presence of ATP (3 μmol/L or 1000 μmol/L), and the phosphorylation level was determined by TR-FRET signal. The mean IC50 was calculated from the concentration-response curve. [1] The kinase selectivity profile of TAK-960 was determined by screening against a panel of 288 kinases using a HotSpot technology platform. Inhibition was measured at a single concentration of TAK-960 (1000 nmol/L). For kinases showing greater than 80% inhibition, full IC50 values were subsequently determined in assays conducted in the presence of 10 μmol/L ATP. [1] |
| Cell Assay |
In 96-well plates, cells are seeded at a density of 3–30 × 103 cells/well using the suitable medium plus 10% fetal calf serum (FCS). The number of viable cells is determined using the CellTiter-Glo Assay 72 hours after the cells are treated with serial dilutions of TAK-960 for 24 hours. Statistical analysis and EC50 value computation are carried out.
For cell proliferation analysis, cancer cell lines were seeded in 96-well plates. After 24 hours, cells were treated with serial dilutions of TAK-960. Following 72 hours of incubation, cell viability was assessed using a luminescent ATP detection assay. The EC50 values for growth inhibition were calculated using appropriate software. [1] For cell cycle analysis, HT-29 cells were seeded, treated with TAK-960 for 48 hours, harvested, and fixed in ethanol. After RNase treatment, cells were stained with propidium iodide, and DNA content was analyzed by flow cytometry to determine the distribution of cells in different cell cycle phases. [1] For immunocytochemistry to assess mitotic spindle morphology, HT-29 cells were treated with TAK-960 for 24 hours, fixed, permeabilized, and blocked. Cells were then incubated with primary antibodies against α-tubulin and γ-tubulin, followed by fluorophore-conjugated secondary antibodies. Nuclei were counterstained, and images were acquired by fluorescence microscopy. [1] For analysis of pHH3 levels in cells, HT-29 cells were treated with TAK-960. After 24 hours, cell lysates were prepared and pHH3 levels were quantified using a specific sandwich ELISA kit according to the manufacturer's instructions. [1] |
| Animal Protocol |
Athymic nude mice (BALB/cAJc1-nu/nu), severe combined immunodeficiency (SCID) mice (C.B17-Icr- scid/scid Jcl) or NOD-scid mice (NOD.CB17-Prkdc scid/J)
30 mg/kg Oral dosing For subcutaneous tumor xenograft models, athymic nude mice or SCID mice were injected subcutaneously with cancer cells (e.g., 5 × 10^6 cells per mouse). When tumor volumes reached approximately 200 mm³, mice were randomized into treatment groups. TAK-960 was suspended in 0.5% methylcellulose and administered orally at specified doses and schedules (e.g., once daily, or intermittent schedules like twice weekly). Tumor volumes and body weights were measured regularly. [1] For the pharmacokinetic/pharmacodynamic (PK/PD) study in HT-29 xenograft-bearing mice, a single oral dose of TAK-960 (5, 10, or 30 mg/kg in 0.5% methylcellulose) was administered. Blood and tumor samples were collected at multiple time points for analysis of drug concentrations and pHH3 levels. [1] For the disseminated leukemia model, NOD/SCID mice were pretreated with an antibody to deplete natural killer cells, sublethally irradiated, and then injected intravenously with MV4-11luc leukemia cells. Treatment with TAK-960 (7.5 mg/kg in 0.5% methylcellulose, orally once daily) or control was initiated. Tumor burden was monitored by in vivo bioluminescence imaging after intraperitoneal injection of the luciferin substrate. [1] For the MDR1-expressing tumor model, SCID mice bearing subcutaneous K562 or K562ADR leukemia xenografts were treated with a single dose of TAK-960 (30 mg/kg, orally) or paclitaxel (20 mg/kg, intraperitoneally). Tumor samples were collected at various time points for immunohistochemical analysis of pHH3-positive cells. [1] |
| ADME/Pharmacokinetics |
In HT-29 tumor-bearing mice, plasma exposure increased dose-proportionately after a single oral dose of TAK-960. The compound preferentially distributed in tumor tissue, with tumor concentrations higher than plasma concentrations at the same time. [1]
The maximum increase in the pharmacodynamic marker pHH3 in tumors occurred 24 hours after administration, at which time the tumor concentration of TAK-960 had reached its peak (between 4 and 8 hours). [1] Total exposure (AUC) of TAK-960 in plasma and tumors was linearly correlated with the administered dose. The peak pharmacodynamic response (Emax of pHH3) was similar at doses of 10 mg/kg and 30 mg/kg, but the change in total pharmacodynamic response over time (AUE) was dose-dependent. [1] |
| Toxicity/Toxicokinetics |
In several xenotransplantation studies, oral effective doses (e.g., 10 mg/kg daily) of TAK-960 did not cause significant weight loss or treatment-related death, indicating good tolerability in these models. [1]
In the K562ADR model, the maximum tolerated dose (MTD) of paclitaxel under the study protocol (10 mg/kg once daily, intraperitoneally, 5 days a week for 2 weeks) has been mentioned, but the specific MTD of TAK-960 has not been provided. [1] |
| References |
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| Additional Infomation |
TAK-960, a PLK1 inhibitor, is an orally potent Polo-like kinase 1 (PLK1) inhibitor with potential antitumor activity. TAK-960 selectively inhibits PLK1, inducing selective G2/M phase cell cycle arrest in various tumor cells, subsequently leading to apoptosis; in normal cells, it causes reversible G1 and G2 phase cell cycle arrest without inducing apoptosis. PLK1 inhibition may suppress the proliferation of PLK1-overexpressing tumor cells. PLK1, named after the Drosophila polo gene, is a serine/threonine kinase that plays a crucial role in the regulation of mitosis. TAK-960 is a novel, orally bioavailable, ATP-competitive PLK1 inhibitor. PLK1 is a serine/threonine kinase essential for mitosis. PLK1 is overexpressed in various cancers and is associated with poor prognosis. [1]
Its mechanism of action involves PLK1 inhibition, leading to mitotic arrest, manifested as accumulation of cells in the G2/M phase, elevated pHH3 levels, and abnormal spindle formation, ultimately inhibiting cancer cell proliferation. [1] Unlike paclitaxel and doxorubicin, TAK-960 remains active against cancer cells that overexpress the multidrug resistance protein MDR1. [1] In mouse models, the antitumor efficacy of TAK-960 is related to the dosing regimen; in the HT-29 model, the daily continuous dosing regimen was more effective than the intermittent dosing regimen. [1] Changes in tumor pHH3 levels can serve as a pharmacodynamic biomarker for TAK-960's inhibition of PLK1 in vitro and in vivo. [1] Based on these preclinical data, TAK-960 entered a Phase I clinical trial for patients with advanced cancer. [1] |
| Molecular Formula |
C27H36CL2F3N7O3
|
|---|---|
| Molecular Weight |
561.59916
|
| Exact Mass |
561.267
|
| Elemental Analysis |
C, 57.74; H, 6.10; F, 10.15; N, 17.46; O, 8.55
|
| CAS # |
1137868-52-0
|
| Related CAS # |
TAK-960 dihydrochloride;TAK-960 hydrochloride;1137868-96-2;TAK-960 monohydrochloride;2108449-45-0
|
| PubChem CID |
53357478
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| Appearance |
White to beige solid powder
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| Density |
1.4±0.1 g/cm3
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| Index of Refraction |
1.615
|
| LogP |
1.33
|
| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
11
|
| Rotatable Bond Count |
6
|
| Heavy Atom Count |
40
|
| Complexity |
903
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
0
|
| InChi Key |
GWRSATNRNFYMDI-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C27H34F3N7O3/c1-35-10-8-16(9-11-35)32-24(38)18-12-22(40-3)20(13-19(18)28)33-26-31-14-21-23(34-26)37(17-6-4-5-7-17)15-27(29,30)25(39)36(21)2/h12-14,16-17H,4-11,15H2,1-3H3,(H,32,38)(H,31,33,34)
|
| Chemical Name |
4-[(9-cyclopentyl-7,7-difluoro-5-methyl-6-oxo-8H-pyrimido[4,5-b][1,4]diazepin-2-yl)amino]-2-fluoro-5-methoxy-N-(1-methylpiperidin-4-yl)benzamide
|
| Synonyms |
TAK 960; TAK960; TAK-960
|
| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
DMSO: 13~16.7 mg/mL (23.2~29.7 mM)
Ethanol: ~3 mg/mL (~5.3 mM) |
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7806 mL | 8.9031 mL | 17.8063 mL | |
| 5 mM | 0.3561 mL | 1.7806 mL | 3.5613 mL | |
| 10 mM | 0.1781 mL | 0.8903 mL | 1.7806 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT01179399 | Terminated | Drug: TAK-960 | Advanced Nonhematological Malignancies |
Millennium Pharmaceuticals, Inc. | September 2010 | Phase 1 |
 TAK-960 is a potent and selective inhibitor of PLK1.Mol Cancer Ther. 2012 Mar;11(3):700-9. |
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 TAK-960 induces accumulation of aberrant mitotic cells in HT-29 cells.Mol Cancer Ther. 2012 Mar;11(3):700-9. |
 PK/PD and antitumor activity analysis for TAK-960 in mice bearing HT-29 colorectal cancer cells.Mol Cancer Ther. 2012 Mar;11(3):700-9. |
 Single-agent efficacy of TAK-960 in various tumor xenograft models using human cancer cell lines.Mol Cancer Ther. 2012 Mar;11(3):700-9. |
|---|
 TAK-960 inhibits proliferation of human cancer cell lines regardless ofTP53andKRASmutation and MDR1 expression status. |
 TAK-960 shows PD activity in paclitaxel-resistant model.Mol Cancer Ther. 2012 Mar;11(3):700-9. |
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