ROS1 (Ki < 0.025 nM); c-Met (IC50 = 11 nM); NPM-ALK (IC50 = 24 nM)

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].

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].

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.

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.

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.

Absorption
In patients with pancreatic cancer, colorectal cancer, sarcoma, anaplastic large cell lymphoma, and non-small cell lung cancer (NSCLC) treated with crizotinib, the mean AUC and Cmax increased proportionally to the dose, ranging from 100 mg once daily to 300 mg twice daily. The median time to peak concentration (tmax) after a single dose of crizotinib was 4 to 6 hours. In patients (n=167) receiving crizotinib 250 mg twice daily multiple times, the mean AUC was 2321.00 ng·hr/mL, the mean Cmax was 99.60 ng/mL, and the median tmax was 5.0 hours. The mean absolute bioavailability of crizotinib was 43%, ranging from 32% to 66%. A high-fat diet reduced the AUC0-INF and Cmax of crizotinib by approximately 14%. Age, sex at birth, and race (Asian vs. non-Asian patients) had no clinically significant effect on the pharmacokinetics of crizotinib. In patients under 18 years of age, higher body weight correlated with lower crizotinib exposure.
Excretion Route
Following a single 250 mg dose of radiolabeled crizotinib in healthy subjects, 63% and 22% of the administered dose were recovered in feces and urine, respectively. Approximately 53% and 2.3% of the administered dose remained unmetabolized in feces and urine, respectively.
Volume of Distribution
The mean volume of distribution (Vss) of crizotinib after a single intravenous dose was 1772 L.
Clearance
At steady state (250 mg twice daily), the mean apparent clearance (CL/F) of crizotinib was 60 L/hr. This value is lower than the clearance (100 L/hr) measured after a single 250 mg oral dose, which may be due to CYP3A autoinhibition.

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Metabolism/Metabolites
Crizotinib is primarily metabolized in the liver via CYP3A4 and CYP3A5, undergoing O-dealkylation followed by a phase II binding reaction. Non-metabolic clearance, such as bile excretion, cannot be ruled out. PF-06260182 (composed of two diastereomers, PF-06270079 and PF-06270080) is currently the only identified active metabolite of crizotinib. In vitro studies have shown that PF-06270079 and PF-06270080 exhibit approximately 3- to 8-fold reduced inhibitory activity against anaplastic lymphoma kinase (ALK) and approximately 2.5- to 4-fold reduced inhibitory activity against hepatocyte growth factor receptor (HGFR, c-Met) compared to crizotinib.

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Biological Half-Life
The terminal plasma half-life of crizotinib after a single dose is 42 hours.

Hepatotoxicity
In early large clinical trials, up to 57% of patients receiving standard-dose crizotinib experienced elevated serum transaminase levels, with 6% having transaminase levels exceeding 5 times the upper limit of normal, leading to premature discontinuation of treatment in 2% to 4%. Elevated serum transaminase levels typically appear 4 to 12 weeks after treatment but are usually not accompanied by jaundice or elevated alkaline phosphatase. Crizotinib can be restarted at a reduced dose after transaminase abnormalities return to normal. Most cases of crizotinib-induced liver injury are mild or asymptomatic and resolve within 1 to 2 months after discontinuation (Case 1). However, there have been reports of jaundice and related symptoms during crizotinib treatment, with 0.1% of these patients dying (Case 2). Severe crizotinib-induced liver injury typically occurs within 2 to 6 weeks of treatment initiation, characterized by significantly elevated serum transaminase levels, followed by jaundice, progressive liver dysfunction, coagulation disorders, hepatic encephalopathy, and death. Therefore, routine liver function tests are recommended every 2 to 4 weeks during treatment. Probability score: C (may lead to clinically significant acute liver injury).

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Use during pregnancy and lactation
◉Overview of use during lactation
There is currently no information on the clinical use of crizotinib during lactation. Because crizotinib binds to plasma proteins at a rate of 91%, its concentration in breast milk may be low. However, its half-life is approximately 42 hours, and it may accumulate in the infant. The manufacturer recommends discontinuing breastfeeding during crizotinib treatment and for 45 days after the last dose.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding rate
Crizotinib binds to plasma proteins at a rate of 91%. In vitro studies have shown that drug concentration does not affect its protein binding rate.

[1]. An orally available small-molecule inhibitor of c-Met, PF-2341066, exhibits cytoreductive antitumor efficacy through antiproliferative and antiangiogenic mechanisms. Cancer Res. 2007, 67(9), 4408-4417.

[2]. Cytoreductive antitumor activity of PF-2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell lymphoma. Mol Cancer Ther. 2007, 6(12 Pt 1), 3314-3322.

[3]. Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK). J Med Chem. 2011 Sep 22;54(18):6342-63.

[4]. Differential (18)F-FDG and 3'-deoxy-3'-(18)F-fluorothymidine PET responses to pharmacologic inhibition of the c-MET receptor in preclinical tumor models. J Nucl Med. 2011 Aug;52(8):1261-7.

c-Met receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) are closely associated with the progression of various human cancers and are highly attractive therapeutic targets. PF-2341066 is a potent, orally bioavailable, ATP-competitive small molecule inhibitor that inhibits the catalytic activity of c-Met kinase. PF-2341066 is selective for c-Met (and anaplastic lymphoma kinase) compared to over 120 different tyrosine and serine/threonine kinases. In vitro experiments showed that PF-2341066 effectively inhibited c-Met phosphorylation and c-Met-dependent proliferation, migration, and invasion of human tumor cells (IC50 values of 5-20 nmol/L). Furthermore, PF-2341066 effectively inhibited HGF-stimulated endothelial cell survival or invasion and serum-stimulated tubular formation in vitro, indicating that the drug also possesses anti-angiogenic properties. In various tumor models expressing activated c-Met, PF-2341066 showed efficacy at well-tolerated doses, including significant cytoreductive antitumor activity. The antitumor efficacy of PF-2341066 was dose-dependent and closely related to the inhibition of c-Met phosphorylation in vivo. The near-maximal inhibition of c-Met activity throughout the dosing interval was a necessary condition for PF-2341066 to exert its maximum efficacy. Other mechanism studies have shown that PF-2341066 can dose-dependently inhibit c-Met-dependent signal transduction, tumor cell proliferation (Ki67), induce apoptosis (caspase-3), and reduce microvessel density (CD31). These results suggest that the antitumor activity of PF-2341066 may be achieved by directly affecting the growth or survival of tumor cells and by anti-angiogenic mechanisms. Overall, these results demonstrate the therapeutic potential of using selective small molecule inhibitors to target c-Met for the treatment of human cancers. [1]

C₂₁H₂₃CL₃FN₅O

486.80

485.095

1415560-69-8

Crizotinib;877399-52-5;Crizotinib-d5;1395950-84-1; 877399-53-6 (acetate)

71576688

Yellow to brown solid

6.749

3

6

5

31

558

1

ClC1=C(C([H])=C([H])C(=C1[C@@]([H])(C([H])([H])[H])OC1=C(N([H])[H])N=C([H])C(=C1[H])C1C([H])=NN(C=1[H])C1([H])C([H])([H])C([H])([H])N([H])C([H])([H])C1([H])[H])Cl)F.Cl[H]

BTDNHKQCPIBABF-UTONKHPSSA-N

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

3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine;hydrochloride

PF-02341066 hydrochloride; PF2341066; PF-2341066; PF-02341066; PF02341066; PF 02341066; PF 2341066; Crizotinib; US trade name: Xalkori

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~97 mg/mL (~199.3 mM)
Ethanol: ~97 mg/mL (~199.3 mM)
Water: ~97 mg/mL (~199.3 mM)

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.

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