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Crizotinib HCl (formerly known as PF-02341066 hydrochloride; trade name: Xalkori) is a potent, orally bioavailable small molecule inhibitor of c-Met and ALK, with IC50 values of 11 nM and 24 nM in assays involving cells, respectively. In the US, crizotinib is licensed for the treatment of non-small cell lung cancer (NSCLC). It is also being investigated in clinical trials for safety and effectiveness in the treatment of neuroblastoma, anaplastic large cell lymphoma, and other advanced solid tumors in both adults and children. Crizotinib inhibits the activation of the MET signaling pathway and the membrane receptor MET, which in susceptible tumor cell populations may prevent tumor cell growth, migration, invasion, and angiogenesis.
| Targets |
ROS1 (Ki < 0.025 nM); c-Met (IC50 = 11 nM); NPM-ALK (IC50 = 24 nM)
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| ln Vitro |
PF-2341066 dexhibits comparable efficacy (IC50 of 5 nM and 20 nM, respectively) against c-Met phosphorylation in mIMCD3 mouse or MDCK canine epithelial cells. When compared to NIH3T3 cells expressing the wild-type receptor, which has an IC50 of 13 nM, PF-2341066 exhibits better or comparable activity against cells engineered to express the c-Met ATP-binding site mutants V1092I or H1094R or the P-loop mutant M1250T, with IC50 values of 19 nM, 2 nM, and 15 nM, respectively. On the other hand, PF-2341066 exhibits a significant change in potency when compared to wild-type receptor when it comes to cells that are engineered to express the c-Met activation loop mutants Y1230C and Y1235D, with IC50 values of 127 nM and 92 nM, respectively. In NCI-H69 and HOP92 cells, which express the endogenous c-Met variants R988C and T1010I, respectively, PF-2341066 also potently inhibits the phosphorylation of c-Met with IC50 values of 13 nM and 16 nM, respectively[1].
PF-2341066 also has an IC50 of 24 nM, which effectively inhibits the phosphorylation of NPM-ALK in Karpas299 or SU-DHL-1 ALCL cells. With an IC50 of 30 nM, PF-2341066 demonstrably inhibits cell proliferation, which is linked to G(1)-S-phase cell cycle arrest and the induction of apoptosis in ALK-positive ALCL cells[2], but not in ALK-negative lymphoma cells[2]. |
| ln Vivo |
PF-2341066 shows that both the 50 mg/kg/day and 75 mg/kg/day treatment cohorts have the potential to cause significant regression of large established tumors (> 600 mm3), with a 60% decrease in mean tumor volume over the 43-day administration schedule in the GTL-16 model. A different study shows that PF-2341066 can completely suppress GTL-16 tumor growth for longer than three months. During the course of the three-month treatment regimen at 50 mg/kg/day, only one out of twelve mice showed a discernible increase in tumor growth. In GTL-16 tumors, there is a notable dose-dependent decrease in CD31-positive endothelial cells at 12.5 mg/kg/day, 25 mg/kg/day, and 50 mg/kg/day. This suggests that MVD inhibition correlates with antitumor efficacy in a dose-dependent manner. In the GTL-16 and U87MG models, PF-2341066 exhibits a notable dose-dependent decrease in human VEGFA and IL-8 plasma levels. Phosphorylated c-Met, Akt, Erk, PLCλ1, and STAT5 levels are markedly inhibited in GTL-16 tumors after PF-2341066 is administered p.o.[1].
Treatment with 50 mg/kg PF-2341066 causes tumor regression in c-MET-amplified GTL-16 xenografts; this tumor regression is accompanied by a gradual decrease in 18F-FDG uptake and a reduction in the expression of GLUT-1, the glucose transporter[4]. |
| Enzyme Assay |
Cells are plated in 96-well plates with 10% fetal bovine serum (FBS)-supplemented media before being switched to serum-free media with 0.04% bovine serum albumin (BSA) after 24 hours. For a maximum of 20 minutes, corresponding growth factors are added in experiments examining ligand-dependent RTK phosphorylation. Following one hour of incubation with PF-2341066 and/or the appropriate ligands for the specified times, cells are once again washed with HBSS supplemented with one milligram of Na3VO4, and protein lysates are produced from the cells. The phosphorylation of particular protein kinases is then measured using a sandwich ELISA technique that employs a detection antibody specific for phosphorylated tyrosine residues and particular capture antibodies used to coat 96-well plates. Antibody-coated plates undergo the following steps: (a) overnight incubation in the presence of protein lysates; (b) seven PBS washes with 1% Tween 20; (c) 30 minutes of incubation in a horseradish peroxidase-conjugated anti-total-phosphotyrosine (PY-20) antibody (1:500); (d) seven more PBS washes; (e) incubation in 3,3′,5,5′-tetramethyl benzidine peroxidase substrate to start a colorimetric reaction that is stopped by adding 0.09 N H2SO4; and (f) absorbance at 450 nm measured with a spectrophotometer.
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| Cell Assay |
After being seeded in 96-well plates with media supplemented with 10% fetal bovine serum (FBS) and 0.04% bovine serum albumin (BSA) for 24 hours, cells, including GTL-16 gastric carcinoma cells and T47D breast carcinoma cells, are moved to serum-free media. For a maximum of 20 minutes, corresponding growth factors are added in experiments examining ligand-dependent RTK phosphorylation. Following an hour of PF-2341066 incubation, or the application of suitable ligands for the specified durations, cells are once again washed with HBSS supplemented with 1 mM Na3VO4, following which protein lysates are extracted from the cells. Then, using a sandwich ELISA technique that employs phosphorylation-specific detection antibodies for phosphorylated tyrosine residues and specific capture antibodies to coat 96-well plates, the phosphorylation of particular protein kinases is evaluated. Antibody-coated plates undergo the following steps: (a) overnight incubation in the presence of protein lysates; (b) seven PBS washes with 1% Tween 20; (c) 30 minutes of incubation in a horseradish peroxidase-conjugated anti-total-phosphotyrosine (PY-20) antibody (1:500); (d) seven more PBS washes; (e) incubation in 3,3′,5,5′-tetramethyl benzidine peroxidase substrate to start a colorimetric reaction that is stopped with 0.09 N H2SO4; and (f) absorbance at 450 nm measured with a spectrophotometer.
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| Animal Protocol |
PF-2341066 is administered orally by gavage to athymic mice carrying xenografts (300-800 mm3) at predetermined dose levels. Mice are humanely put to sleep at predetermined intervals after PF-2341066 administration, and tumors are removed. Using a liquid nitrogen-cooled cryomortar and pestle, tumors are snap frozen, ground into a paste, protein lysates are produced, and protein concentrations are measured with a BSA assay. Through the use of immunoprecipitation-immunoblotting or capture ELISA, the amount of total and phosphorylated protein is measured.
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| References |
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| Molecular Formula |
C₂₁H₂₃CL₃FN₅O
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|---|---|
| Molecular Weight |
486.80
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| Exact Mass |
485.095222
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| CAS # |
1415560-69-8
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| Related CAS # |
Crizotinib;877399-52-5;Crizotinib-d5;1395950-84-1
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| Appearance |
Solid
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| SMILES |
C[C@H](C1=C(C=CC(=C1Cl)F)Cl)OC2=C(N=CC(=C2)C3=CN(N=C3)C4CCNCC4)N.Cl
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| InChi Key |
BTDNHKQCPIBABF-UTONKHPSSA-N
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| InChi Code |
InChI=1S/C21H22Cl2FN5O.ClH/c1-12(19-16(22)2-3-17(24)20(19)23)30-18-8-13(9-27-21(18)25)14-10-28-29(11-14)15-4-6-26-7-5-15;/h2-3,8-12,15,26H,4-7H2,1H3,(H2,25,27);1H/t12-;/m1./s1
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| Chemical Name |
3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine;hydrochloride
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| Synonyms |
PF-02341066 hydrochloride; PF2341066; PF-2341066; PF-02341066; PF02341066; PF 02341066; PF 2341066; Crizotinib; US trade name: Xalkori
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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: ~97 mg/mL (~199.3 mM)
Ethanol: ~97 mg/mL (~199.3 mM) Water: ~97 mg/mL (~199.3 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 55 mg/mL (112.98 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
(Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0542 mL | 10.2712 mL | 20.5423 mL | |
| 5 mM | 0.4108 mL | 2.0542 mL | 4.1085 mL | |
| 10 mM | 0.2054 mL | 1.0271 mL | 2.0542 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.
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