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].

In 96-well plates, cells are seeded with media supplemented with 10% fetal bovine serum (FBS) and, after 24 hours, are transferred to serum-free media containing 0.04% bovine serum albumin (BSA). Related growth factors are added for up to 20 minutes in experiments looking into ligand-dependent RTK phosphorylation. Protein lysates are produced from cells after they are incubated with PF-2341066 for one hour and/or the appropriate ligands for the specified times. The cells are then once again washed with HBSS supplemented with one milligram of Na3VO4. After that, phosphorylation of particular protein kinases is evaluated by sandwich ELISA technique, which employs a detection antibody specific for phosphorylated tyrosine residues and specific capture antibodies used to coat 96-well plates. Protein lysates are added to antibody-coated plates and incubated for one night at 4°C. Next, the plates are rinsed seven times in 1% Tween 20 in PBS, then incubated for thirty minutes in a horseradish peroxidase-conjugated anti-total-phosphotyrosine (PY-20) antibody (1:500). Finally, the plates are rinsed seven times more. Finally, the plates are incubated 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 using a spectrophotometer.

In low density, tumor cells are seeded in 96-well plates with growth media supplemented with 10% FBS.After 24 hours, the cells are moved to serum-free media containing 0.04% BSA and 0% FBS. Each well is filled with the appropriate controls or specified concentrations of PF-2341066, and the cells are incubated for a duration of 24 to 72 hours. After being seeded in 96-well plates with EGM2 media for 5 to 6 hours at a density of over 20,000 cells per well, human umbilical vascular endothelial cells (HUVEC) are overnight moved to serum-free medium. The next day, each well is filled with the appropriate controls or designated concentrations of PF-2341066. Following a one-hour incubation period, 100 ng/mL of HGF is added to the designated wells. To ascertain the relative tumor cell or HUVEC, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay is conducted.

PF-2341066 is administered orally by gavage to athymic mice carrying xenografts (300-800 mm3) at predetermined dose levels. Mice are given PF-2341066 at predetermined intervals, and tumors are removed with humane care. 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, Distribution and Excretion
In patients with pancreatic cancer, colorectal cancer, sarcoma, anaplastic large cell lymphoma, and non-small cell lung cancer (NSCLC) receiving crizotinib, the mean AUC and Cmax increased proportionally to the dose, ranging from 100 mg once daily to 300 mg twice daily. The median 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 can reduce 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 was associated with lower crizotinib exposure. 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. The mean volume of distribution (Vss) of crizotinib after a single intravenous administration was 1772 L. 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 pathways, such as biliary 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 after a single dose of crizotinib 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 five 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 treatment can be restarted at a lower dose once 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, 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).
Pregnancy and Lactation Effects
◉ 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, which may allow it to 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.
Protein Binding
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.

Pharmacodynamics
In a phase I study, 37 patients with refractory to multiple solid tumors received crizotinib at 50 to 300 mg daily or twice daily. Two patients with non-small cell lung cancer (NSCLC) harboring echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) mutations responded to treatment; therefore, subsequent studies focused on advanced ALK-positive patients. In this group, the 6-month progression-free survival rate in the crizotinib treatment group was approximately 72%. The two-year overall survival rate was significantly higher in ALK-mutant positive patients receiving crizotinib compared to those who did not receive crizotinib (54% vs 36%). Crizotinib use may cause hepatotoxicity, interstitial lung disease (ILD), pneumonia, QT interval prolongation, bradycardia, severe vision loss, embryo-fetal toxicity, and gastrointestinal toxicity in children and adolescents with anaplastic large cell lymphoma (ALCL) or inflammatory myofibroblastic tumor (IMT).

C23H26CL2FN5O3

510.3914

509.14

C, 54.13; H, 5.13; Cl, 13.89; F, 3.72; N, 13.72; O, 9.40

877399-53-6

Crizotinib-d5;1395950-84-1; Crizotinib hydrochloride;1415560-69-8;Crizotinib-d5;1395950-84-1; 877399-52-5

11626560

Typically exists as solid at room temperature

6.038

2

6

5

30

558

1

ClC1C(=CC=C(C=1[C@@H](C)OC1=C(N)N=CC(=C1)C1C=NN(C=1)C1CCNCC1)Cl)F

LFCVDLCLKZRGFW-UTONKHPSSA-N

InChI=1S/C21H22Cl2FN5O.C2H4O2/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;1-2(3)4/h2-3,8-12,15,26H,4-7H2,1H3,(H2,25,27);1H3,(H,3,4)/t12-;/m1./s1

(R)-3-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine acetate

PF 2341066; PF-2341066; Crizotinib (acetate); 2-Pyridinamine, 3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-[1-(4-piperidinyl)-1H-pyrazol-4-yl]-, acetate (1:1); FB99MH8KWZ; SCHEMBL1827232; DTXSID00236600; LFCVDLCLKZRGFW-UTONKHPSSA-N; PF2341066

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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