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, colorectal, sarcoma, anaplastic large-cell lymphoma and non-small cell lung cancer (NSCLC) treated with crizotinib doses ranging from 100 mg once a day to 300 mg twice a day, the mean AUC and Cmax increased in a dose-proportional manner. A single crizotinib dose of crizotinib is absorbed with a median tmax 4 to 6 hours. In patients receiving multiple doses of crizotinib 250 mg twice daily (n=167), the mean AUC was is 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 is 43%, ranging from 32% to 66%. High-fat meals reduce the AUC0-INF and Cmax of crizotinib by approximately 14%. Age, sex at birth, and ethnicity (Asian vs non-Asian patients) did not have a clinically significant effect on crizotinib pharmacokinetics. In patients less than 18 years old, higher body weight was associated with a lower crizotinib exposure.

Route of Elimination
After administering a single 250 mg radiolabeled crizotinib dose to healthy subjects, 63% and 22% of the administered dose were recovered in feces and urine. Unchanged crizotinib represented approximately 53% and 2.3% of the administered dose in feces and urine, respectively.

Volume of Distribution
Following a single intravenous dose, the mean volume of distribution (Vss) of crizotinib was 1772 L.

Clearance
At steady-state (250 mg twice daily), crizotinib has a mean apparent clearance (CL/F) of 60 L/hr. This value is lower than the one detected after a single 250 mg oral dose (100 L/hr),, possibly due to CYP3A auto-inhibition.

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Metabolism / Metabolites
Crizotinib is mainly metabolized in the liver by CYP3A4 and CYP3A5, and undergoes an O-dealkylation, with subsequent phase 2 conjugation. Non-metabolic elimination, such as biliary excretion, can not be excluded. PF-06260182 (with two constituent diastereomers, PF-06270079 and PF-06270080) is the only active metabolite of crizotinib that has been identified. _In vitro_ studies suggest that, compared to crizotinib, PF-06270079 and PF-06270080 are approximately 3- to 8-fold less potent against anaplastic lymphoma kinase (ALK) and 2.5- to 4-fold less potent against Hepatocyte Growth Factor Receptor (HGFR, c-Met).

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Biological Half-Life
Following single doses of crizotinib, the plasma terminal half-life was 42 hours.

Hepatotoxicity
In large early clinical trials, elevations in serum aminotransferase levels occurred in up to 57% of patients treated with standard doses of crizotinib, were greater than 5 times ULN in 6% of patients, and led to early discontinuation of therapy in 2% to 4% of patients. Serum aminotransferase elevations typically arose after 4 to 12 weeks of treatment, but usually without jaundice or alkaline phosphatase elevations. Restarting crizotinib after resolution of the aminotransferase abnormalities can be done starting with a reduced dose. Most cases of liver injury due to crizotinib have been minimally or not symptomatic, and the injury resolved within 1 to 2 months of stopping the drug (Case 1). However, cases with jaundice and symptoms during crizotinib therapy have been reported which were fatal in 0.1% of treated patients (Case 2). The severe cases of liver injury due to crizotinib typically arose within 2 to 6 weeks of starting therapy and presented with marked elevations in serum aminotransferase levels followed by jaundice, progressive hepatic dysfunction, coagulopathy, encephalopathy and death. For these reasons, routine periodic monitoring of liver tests at 2 to 4 week intervals during therapy is recommended. Likelihood score: C (probable cause of clinically apparent acute liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of crizotinib during breastfeeding. Because crizotinib is 91% bound to plasma proteins, the amount in milk is likely to be low. However, its half-life is about 42 hours and it might accumulate in the infant. The manufacturer recommends that breastfeeding be discontinued during crizotinib therapy and for 45 days after the last dose.

◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.

◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Crizotinib is 91% bound to plasma protein. _In vitro_ studies suggest that this is not affected by drug concentration.

[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.

The c-Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), have been implicated in the progression of several human cancers and are attractive therapeutic targets. PF-2341066 was identified as a potent, orally bioavailable, ATP-competitive small-molecule inhibitor of the catalytic activity of c-Met kinase. PF-2341066 was selective for c-Met (and anaplastic lymphoma kinase) compared with a panel of >120 diverse tyrosine and serine-threonine kinases. PF-2341066 potently inhibited c-Met phosphorylation and c-Met-dependent proliferation, migration, or invasion of human tumor cells in vitro (IC(50) values, 5-20 nmol/L). In addition, PF-2341066 potently inhibited HGF-stimulated endothelial cell survival or invasion and serum-stimulated tubulogenesis in vitro, suggesting that this agent also exhibits antiangiogenic properties. PF-2341066 showed efficacy at well-tolerated doses, including marked cytoreductive antitumor activity, in several tumor models that expressed activated c-Met. The antitumor efficacy of PF-2341066 was dose dependent and showed a strong correlation to inhibition of c-Met phosphorylation in vivo. Near-maximal inhibition of c-Met activity for the full dosing interval was necessary to maximize the efficacy of PF-2341066. Additional mechanism-of-action studies showed dose-dependent inhibition of c-Met-dependent signal transduction, tumor cell proliferation (Ki67), induction of apoptosis (caspase-3), and reduction of microvessel density (CD31). These results indicated that the antitumor activity of PF-2341066 may be mediated by direct effects on tumor cell growth or survival as well as antiangiogenic mechanisms. Collectively, these results show the therapeutic potential of targeting c-Met with selective small-molecule inhibitors 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.)