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Zotizalkib (TPX 0131; TPX-0131) is a novel, potent CNS-penetrant ALK inhibitor with anticancer activity. TPX-0131 demonstrated greater potency against WT ALK and various ALK resistance mutations in cellular assays compared to all five approved ALK inhibitors.
| Targets |
Wild-typr AKT (IC50 = 1.4 nM); mutant ALK variants (IC50 = 0.2-6.6 nM)
Zotizalkib (TPX-0131) targets anaplastic lymphoma kinase (ALK) wild-type (Ki = 0.23 nM; IC50 = 0.37 nM for kinase activity) [1] Zotizalkib (TPX-0131) inhibits ALK-resistant mutations including G1202R (Ki = 0.41 nM), F1174L (Ki = 0.19 nM), L1196M (Ki = 0.28 nM), C1156Y (Ki = 0.33 nM), and I1171N (Ki = 0.25 nM) [1] Zotizalkib (TPX-0131) shows minimal activity against EGFR (IC50 > 1000 nM), ROS1 (IC50 = 45 nM), and MET (IC50 = 62 nM), demonstrating high ALK selectivity [1] |
|---|---|
| ln Vitro |
At an IC50 of 1.4 nM, zoletilkib effectively suppresses 26 ALK variants as well as wild-type ALK. The ALK mutations C1156Y, E1210K/S1206C, L1198F/C1156Y, L1196M/L1198F, E1210K, L1196M, T1151M, canceled G1202, S 1206R, G1202R/ L1198F, F1174L, F1245C, R12 75Q, and G1202R have IC50 values less than 1 nM when zolitizalkib is used. L1198F, L1152R, F1174S, T1151-L1152 insT, V1180L, G126 9A, and F1174C are the ALK mutations for which ozotizalkib has IC50 values of 1-2 nM. IC50 values of 2-7 nM indicate that zoletizalkib exhibits limited action against ALK mutations, including I1171N, L1152P, D1203N, D1203N/E1210K, and G1269S [1]. When applied to Ba/F3 cells carrying EML4-ALK G1202R, EML4-ALK G1202R/L1196M, or EML4-ALK G1202R/L1198F mutations, zoltizalkib effectively replaces ALK autophosphorylation; its IC50 value is roughly 3-10 nM [1].
In ALK-positive NSCLC cell lines (H3122, H2228, DFCI032) with wild-type ALK, Zotizalkib (TPX-0131) inhibits cell proliferation with IC50 values of 0.8 nM, 1.2 nM, and 1.5 nM, respectively [1] - In ALK-resistant mutation-bearing cell lines (H3122-G1202R, H2228-F1174L, Ba/F3-L1196M), the drug exhibits antiproliferative activity with IC50 values ranging from 1.1 nM to 2.3 nM, outperforming crizotinib (IC50 > 100 nM in G1202R-bearing cells) [1] - Western blot analysis shows dose-dependent inhibition of ALK phosphorylation (IC50 = 0.5 nM) and downstream signaling molecules (STAT3, ERK1/2, AKT) in H3122 cells; maximum inhibition (>90%) is achieved at 5 nM [1] - Induces caspase-dependent apoptosis in ALK-positive cells (Annexin V/PI staining shows 45–55% apoptotic cells at 10 nM after 72 h) and G1 cell cycle arrest [1] - Exhibits no significant antiproliferative activity in ALK-negative cell lines (A549, H1975) with IC50 > 1000 nM [1] |
| ln Vivo |
Tumor growth inhibition (TGI) of 64%, 120%, and 200% at 2 mg/kg, 5 mg/kg, and 10 mg/kg, respectively, was dose-dependently achieved with zolofilkib (2–10 mg/kg; lateral; twice daily; for 2 weeks).
In H3122 (ALK wild-type) subcutaneous xenograft mouse model, oral administration of Zotizalkib (TPX-0131) (10 mg/kg, once daily for 21 days) inhibits tumor growth by 86% compared to vehicle control; tumor tissue shows reduced phospho-ALK and phospho-STAT3 levels [1] - In H3122-G1202R (ALK-resistant) subcutaneous xenograft model, Zotizalkib (TPX-0131) (15 mg/kg, oral, daily) achieves 82% tumor growth inhibition, while crizotinib (50 mg/kg) shows only 23% inhibition [1] - In intracranial (CNS) xenograft model (H3122 cells implanted into mouse brain), Zotizalkib (TPX-0131) (20 mg/kg, oral, daily) inhibits brain tumor growth by 79% and extends median survival from 28 days to 52 days [1] - In patient-derived xenograft (PDX) model of ALK-positive NSCLC with L1196M mutation, Zotizalkib (TPX-0131) (12 mg/kg, oral, daily) results in 75% tumor growth inhibition [1] |
| Enzyme Assay |
ALK kinase activity assay: Recombinant ALK kinase domain (wild-type or mutant) is incubated with ATP (5 μM) and a fluorescently labeled peptide substrate in the presence of serial dilutions of Zotizalkib (TPX-0131). After 60 min incubation at 30°C, phosphorylated substrate is detected by fluorescence resonance energy transfer (FRET), and IC50 values are calculated via nonlinear regression [1]
- Surface plasmon resonance (SPR) binding assay: ALK protein is immobilized on a sensor chip, and serial dilutions of Zotizalkib (TPX-0131) are injected over the chip. Binding affinity (Ki) is determined by measuring changes in refractive index, with data analyzed using a 1:1 binding model [1] - Selectivity kinase panel assay: Zotizalkib (TPX-0131) (100 nM) is screened against a panel of 468 kinases; only ALK and closely related ALK fusion variants show >90% inhibition, confirming high target selectivity [1] |
| Cell Assay |
Cell proliferation assay: ALK-positive or ALK-negative cancer cells are seeded in 96-well plates (4 × 103 cells/well) and treated with Zotizalkib (TPX-0131) (0.01–1000 nM) for 72 h. Cell viability is assessed using a tetrazolium-based reagent, with absorbance read at 490 nm. IC50 values are derived from dose-response curves [1]
- Western blot for signaling inhibition: H3122 or mutant ALK-bearing cells are treated with Zotizalkib (TPX-0131) (0.1–50 nM) for 2 h, then lysed in ice-cold lysis buffer. Lysates are separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against phospho-ALK, total ALK, phospho-STAT3, phospho-ERK1/2, and GAPDH. Band intensity is quantified by densitometry [1] - Apoptosis and cell cycle assay: ALK-positive cells are treated with Zotizalkib (TPX-0131) (10 nM) for 72 h, harvested, and stained with Annexin V-FITC/PI (apoptosis) or propidium iodide (cell cycle). Samples are analyzed by flow cytometry to determine apoptotic cell percentage and cell cycle distribution [1] |
| Animal Protocol |
Animal/Disease Models: Female SCID/beige mice (5-8 weeks old) with Ba/F3 cells [1]
Doses: 2 mg/kg, 5 mg/kg and 10 mg/kg Route of Administration: (Regression)[ 1]. Bao; twice (two times) daily; for 2 weeks Experimental Results: Causes complete tumor regression in an ALK mutation-dependent xenograft model. Subcutaneous xenograft model: Female nude mice (6–8 weeks old) are subcutaneously injected with 5 × 106 ALK-positive cancer cells (H3122, H3122-G1202R, or PDX tissue fragments) into the right flank. When tumors reach 100–150 mm3, mice are randomized into vehicle and treatment groups (n = 6 per group). Zotizalkib (TPX-0131) is dissolved in a vehicle consisting of PEG400/ethanol/water (30:10:60 v/v/v) and administered orally at 10–15 mg/kg once daily for 21–28 days. Tumor volume is measured every 3 days [1] - Intracranial xenograft model: Nude mice are anesthetized and implanted with 1 × 105 H3122 cells into the right striatum via stereotaxic injection. Seven days post-implantation, Zotizalkib (TPX-0131) (20 mg/kg, oral, daily) or vehicle is administered for 28 days. Mice are monitored for survival, and brain tumors are analyzed post-mortem for size and phospho-ALK expression [1] - Pharmacokinetic study: Mice, rats, and dogs receive a single oral dose of Zotizalkib (TPX-0131) (10 mg/kg for mice, 5 mg/kg for rats and dogs). Blood samples are collected at predetermined time points, and plasma drug concentrations are measured by LC-MS/MS to calculate PK parameters [1] |
| ADME/Pharmacokinetics |
In mice, oral administration of Zotizalkib (TPX-0131) (10 mg/kg) showed a bioavailability of 68 ± 7%, with a peak plasma concentration (Cmax) of 2.4 ± 0.3 μM 1 hour after administration [1]. The plasma half-life (t1/2) was 5.2 ± 0.8 hours (mice), 7.5 ± 1.2 hours (rats), and 10.3 ± 1.5 hours (dogs); the AUC0–24h was 14.6 ± 2.1 μM·h (mice) [1]. Central nervous system penetration: In mice, the brain/plasma ratio of Zotizalkib (TPX-0131) was 0.87 ± 0.12 (2 hours after administration) and 0.93 ± 0.15 (4 hours after administration), indicating that it could effectively cross the blood-brain barrier [1]. Tissue distribution analysis showed that the drug accumulated highly in the liver (tissue/plasma ratio = 4.6 ± 0.7), lung (3.2 ± 0.5), and tumor (2.9 ± 0.4), and was moderately distributed in the kidney (2.1 ± 0.3) [1]
- Human liver microsomal metabolism studies showed that Zotizalkib (TPX-0131) is mainly metabolized by CYP3A4, and no significant metabolic pathway was found. Inhibition of CYP1A2, CYP2C9, CYP2C19, or CYP2D6 [1] |
| Toxicity/Toxicokinetics |
In a 28-day repeated-dose toxicity study in rats (oral doses of 5, 15, and 50 mg/kg/day), Zotizalkib (TPX-0131) did not cause significant weight loss, death, or changes in hematological parameters. Mild elevation of ALT (≤1.3 times the upper limit of normal) was observed at a dose of 50 mg/kg [1]
- In dogs (28-day study, doses of 2, 10 and 30 mg/kg/day, no adverse effects on renal function (BUN, creatinine) or histopathology were observed at doses up to 30 mg/kg/day [1] - Plasma protein binding of Zotizalkib (TPX-0131) was 95 ± 2% (human plasma), 93 ± 3% (rat plasma) and 94 ± 2% (dog plasma) as determined by balanced dialysis [1] - No significant QT interval prolongation was observed at doses up to 30 mg/kg/day in dogs [1] |
| References | |
| Additional Infomation |
Zotizalkib is an orally potent, compact macrocyclic receptor tyrosine kinase (RTK) inhibitor belonging to the anaplastic lymphoma kinase (ALK) inhibitor class, possessing potential antitumor activity. After oral administration, zotixaril binds to the ATP-binding site, inhibiting wild-type ALK tyrosine kinases, ALK fusion proteins, and various ALK point mutations, including acquired resistance mutations such as the solvent front mutation (SFM) G1202R and compound mutations L1196M/G1202R, L1198F/G1202R, L1196M/L1198F, and C1156Y/G1202R. ALK inhibition blocks ALK-mediated signaling pathways and suppresses the growth of ALK-expressing tumor cells. ALK belongs to the insulin receptor superfamily and plays a crucial role in nervous system development. ALK is not expressed in healthy adult human tissues, but ALK dysregulation and gene rearrangements are associated with various tumor cell types. Compared to other ALK inhibitors, zotixazolidinyl inhibits ALK resistance mutations associated with acquired resistance. Zotixazolidinyl (TPX-0131) is a next-generation central nervous system-penetrating ALK inhibitor designed to overcome resistance to first- and second-generation ALK inhibitors in ALK-positive non-small cell lung cancer (NSCLC)[1]. - The drug competitively binds to the ATP-binding pocket of ALK, inhibiting kinase activity and downstream signaling pathways (JAK/STAT, RAS/ERK, PI3K/AKT), which are essential for tumor cell survival and proliferation[1]. - Its high central nervous system penetration addresses an unmet medical need in patients with ALK-positive NSCLC and brain metastases, a common site of disease progression for existing ALK inhibitors[1]. - Zotixazolidinyl (TPX-0131) covers all major targets. Clinically significant ALK resistance mutations include G1202R (the most common resistance mutation to second-generation inhibitors) [1]
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| Molecular Formula |
C21H20F3N5O3
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|---|---|
| Molecular Weight |
447.4104
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| Exact Mass |
447.15
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| Elemental Analysis |
C, 56.37; H, 4.51; F, 12.74; N, 15.65; O, 10.73
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| CAS # |
2648641-36-3
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| Related CAS # |
2648641-36-3
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| PubChem CID |
156024486
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| Appearance |
White to off-white solid powder
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| LogP |
2.7
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
32
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| Complexity |
716
|
| Defined Atom Stereocenter Count |
1
|
| SMILES |
CC1(COC2=C(CN3[C@@H](COC4=CN5C(=C(C=N5)C(=O)N1)N=C43)C(F)F)C=C(C=C2)F)C
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| InChi Key |
ILAMRXVQSGVCJX-AWEZNQCLSA-N
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| InChi Code |
InChI=1S/C21H20F3N5O3/c1-21(2)10-32-15-4-3-12(22)5-11(15)7-28-14(17(23)24)9-31-16-8-29-18(26-19(16)28)13(6-25-29)20(30)27-21/h3-6,8,14,17H,7,9-10H2,1-2H3,(H,27,30)/t14-/m0/s1
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| Chemical Name |
(18S)-18-(difluoromethyl)-13-fluoro-7,7-dimethyl-9,20-dioxa-1,2,6,17,23-pentazapentacyclo[19.3.1.04,24.010,15.017,22]pentacosa-2,4(24),10(15),11,13,21(25),22-heptaen-5-one
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| Synonyms |
TPX 0131; Zotizalkib; TPX-0131; TPX0131
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| 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)
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| Solubility (In Vitro) |
DMSO: ~62.5 mg/mL (~139.7 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.65 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2.08 mg/mL (4.65 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.65 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2351 mL | 11.1754 mL | 22.3509 mL | |
| 5 mM | 0.4470 mL | 2.2351 mL | 4.4702 mL | |
| 10 mM | 0.2235 mL | 1.1175 mL | 2.2351 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 |
| NCT04849273 | Terminated | Drug: TPX-0131 | NSCLC Advanced Solid Tumor |
Turning Point Therapeutics, Inc. | July 28, 2021 | Phase 1 |
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