EGFR (IC50 = 6 nM); ErbB2 (IC50 = 45.7 nM); ErbB4 (IC50 = 73.7 nM)

Dacomitinib is administered to nu/nu mice via xenografts created from HCC827 GFP and HCC827 Del/T790M cells in order to assess the drug's effectiveness. The growth of HCC827 GFP xenografts is effectively inhibited by dacomitinib (10 mg/kg/d by daily oral gavage). On the other hand, ZD1839 cannot be used on HCC827 Del/T790M xenografts, and dacomitinib treatment significantly inhibits the growth of this xenograft model[1].

The ERBB1 sequence (Met-668 to Ala-1211), ERBB2 sequence (Ile-675 to Val-1256), and ERBB4 sequence (Gly-259 to Gly-690) are cloned using PCR into the baculoviral vector pFastBac to create the ERBB1, ERBB2, and ERBB4 cytoplasmic fusion proteins. In Sf9 insect cells infected with baculovirus, proteins are expressed as GST fusion proteins. Glutathione sepharose beads are used in affinity chromatography to purify the proteins. An ELISA-based receptor tyrosine kinase assay is used to measure inhibition of ERBB tyrosine kinase activity. In 96-well plates coated with 0.25 mg/mL poly-Glu-Tyr, kinase reactions are conducted with the following conditions: 50 mM HEPES, pH 7.4, 125 mM NaCl, 10 mM MgCl₂, 100 μM sodium orthovanadate, 2 mM dithiothreitol, 20 μM ATP, PF299804 or vehicle control, and 1–5 nM GST-erbB per 50 μL of reaction mixture. The reactions are shaken and allowed to incubate for six minutes at room temperature. After removing the reaction mixture to halt the kinase reactions, the wells are cleaned using wash buffer (0.1% Tween 20 in PBS). The detection of phosphorylated tyrosine residues involves the addition of 0.2 μg/mL antiphosphotyrosine antibody (Oncogene Ab-4; 50 μL/well) in combination with diluted horseradish peroxidase (HRP) in PBS containing 3% BSA and 0.05% Tween 20. The mixture is then shaken at room temperature for 25 minutes. After the antibody is eliminated, wash buffer is used to wash the plates. 50 μL of the HRP substrate (SureBlue3,3,5,5-tetramethyl benzidine, or TMB) is added to each well, and it is shaken at room temperature for 10 to 20 minutes of incubation. 50 μL of the stop solution (0.09 N H₂SO₄) is added to the TMB reaction to halt it. The absorbance at 450 nm is used to quantify the signal. The median effect method is utilized to ascertain the IC50 values for PF299804.

In 24-well plates, duplicate cells are seeded at 5×10³ to 5×10⁴ cells per well, and growth inhibition data is computed. In short, a dose-response curve is generated by adding Dacomitinib at 10 μM and performing 2-fold dilutions over a range of 12 concentrations the day after plating. Also seeded are control wells devoid of the medication. The cells are counted when the drug is added on day 1 and again when the experiment is over, which is six days later. Using a Coulter Z1 particle counter, cells are counted as soon as they are placed in an isotone solution following trypsinization. Using a Coulter Vi-Cell counter, the suspension cultures are tallied[2].

Absorption, Distribution and Excretion
Dacomitinib has shown a linear kinetics after single and multiple dose range studies. The absorption and distribution do not seem to be affected by food or the consumption of antacids. The peak plasma concentration after a dosage of 45 mg for 4 days is of 104 ng/ml. The reported AUC0-24h and tmax are of 2213 ng.h/mL and 6 hours, respectively. As well, following oral administration, the absolute oral bioavailability is 80%.
From the administered dose, 79% is recovered in feces, from which 20% represents the unmodified form of dacomitinib, and 3% is recovered in urine, from which <1% is represented by the unchanged form.
The volume of distribution of dacomitinib was reported to be of 2415 L.
The geometric apparent clearance of dacomitinib is 27.06 L/h.

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Metabolism / Metabolites
Dacomitinib presents an oxidative and conjugative metabolism marked mainly by the activity of glutathione and cytochrome P450 enzymes. After metabolism, its major circulating metabolite is an O-desmethyl dacomitinib form named PF-05199265. This metabolite has been shown to be formed by an oxidative step by CYP2D6 and to a smaller extent by CYP2C9. The following steps of the metabolism are mainly mediated by CYP3A4 for the formation of smaller metabolites. From these metabolic studies, it was shown that dacomitinib inhibited strongly the activities of CYP2D6.

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Biological Half-Life
Dacomitinib is reported to have a very large half-life of 70 hours.

Hepatotoxicity
In large early clinical trials, elevations in serum aminotransferase levels were common during dacomitinib therapy, arising in 40% of patients treated with standard doses. However, most elevations were transient and asymptomatic, and they rarely led to dose modification or discontinuation. Serum ALT elevations above 5 times the ULN occurred in only 1.4% of patients, these rates being lower than with other EGRF inhibitors such as erlotinib and gefitinib. Serum alkaline phosphatase elevations also occurred but were not common. There were no instances of clinically apparent liver injury with jaundice. However, clinical experience with dacomitinib has been limited.
Likelihood score: E* (unproven but suspected rare cause of clinically apparent 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 dacomitinib during breastfeeding. Because dacomitinib is 98% bound to plasma proteins, the amount in milk is likely to be low. However, because of its potential toxicity in the breastfed infant and its half-life of 70 hours, the manufacturer recommends that breastfeeding be discontinued during dacomitinib therapy and for at least 17 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
Dacomitinib is known to present a protein binding of 98%.

[1]. PF00299804, an irreversible pan-ERBB inhibitor, is effective in lung cancer models with EGFR and ERBB2 mutations that are resistant to ZD1839. Cancer Res. 2007 Dec 15;67(24):11924-32.

[2]. Dacomitinib (PF-00299804), an irreversible Pan-HER inhibitor, inhibits proliferation of HER2-amplified breast cancer cell lines resistant to Anti-Human HER2 and GW572016. Mol Cancer Ther. 2012 Sep;11(9):1978-87.

Dacomitinib is a member of the class of quinazolines that is 7-methoxyquinazoline-4,6-diamine in which the amino group at position 4 is substituted by a 3-chloro-4-fluorophenyl group and the amino group at position 6 is substituted by an (E)-4-(piperidin-1-yl)but-2-enoyl group. It has a role as an epidermal growth factor receptor antagonist and an antineoplastic agent. It is a member of quinazolines, a member of piperidines, an enamide, a member of monochlorobenzenes, a member of monofluorobenzenes, a tertiary amino compound, a secondary amino compound and a secondary carboxamide.
Dacomitinib, designed as (2E)-N-16-4-(piperidin-1-yl) but-2-enamide, is an oral highly selective quinazalone part of the second-generation tyrosine kinase inhibitors which are characterized by the irreversible binding at the ATP domain of the epidermal growth factor receptor family kinase domains. Dacomitinib was developed by Pfizer Inc and approved by the FDA on September 27, 2018. Some evidence in the literature suggests the therapeutic potential of dacomitinib in the epithelial ovarian cancer model, although further investigations are needed.
Dacomitinib is a multi-kinase receptor inhibitor used in the therapy of cases of non-small cell lung cancer that harbor activating mutations in the epidermal growth factor receptor gene (EGFR). Dacomitinib is associated with high rate of transient serum aminotransferase elevations during therapy but has not been linked to instances of clinically apparent acute liver injury.
Dacomitinib is a highly selective, orally bioavailable small-molecule inhibitor of the HER family of tyrosine kinases with potential antineoplastic activity. Dacomitinib specifically and irreversibly binds to and inhibits human Her-1, Her-2, and Her-4, resulting in the proliferation inhibition and apoptosis of tumor cells that overexpress these receptors.

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Drug Indication
Dacomitinib is indicated as the first-line treatment of patients with metastatic non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) exon 19 deletion or exon 21 L858R substitution mutations as verified by an FDA-approved test. Lung cancer is the leading cause of cancer death and NSCLC accounts for 85% of lung cancer cases. From the cases of NSCLC, approximately 75% of the patients present a late diagnosis with metastatic and advanced disease which produces a survival rate of 5%. The presence of a mutation in EGFR accounts for more than the 60% of the NSCLC cases and the overexpression of EGFR is associated with frequent lymph node metastasis and poor chemosensitivity.
FDA Label
Vizimpro, as monotherapy, is indicated for the first-line treatment of adult patients with locally advanced or metastatic non small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) activating mutations.

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Mechanism of Action
Dacomitinib is an irreversible small molecule inhibitor of the activity of the human epidermal growth factor receptor (EGFR) family (EGFR/HER1, HER2, and HER4) tyrosine kinases. It achieves irreversible inhibition via covalent bonding to the cysteine residues in the catalytic domains of the HER receptors. The affinity of dacomitinib has been shown to have an IC50 of 6 nmol/L. The ErbB or epidermal growth factor (EGF) family plays a role in tumor growth, metastasis, and treatment resistance by activating downstream signal transduction pathways such as such as Ras-Raf-MAPK, PLCgamma-PKC-NFkB and PI3K/AKT through the tyrosine kinase-driven phosphorylation at the carboxy-terminus. Around 40% of cases show amplification of EGFR gene and 50% of the cases present the _EGFRvIII_ mutation which represents a deletion that produces a continuous activation of the tyrosine kinase domain of the receptor.

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Pharmacodynamics
Preclinical data suggested that dacomitinib increases the inhibition of the epidermal growth factor receptor kinase domain as well as the activity in cell lines harboring resistance mutations such as T790M. This activity further produced a significant reduction of EGFR phosphorylation and cell viability. In these studies, non-small cell lymphoma cancer cell lines with L858R/T790M mutations where used and an IC50 of about 280 nmol/L was observed. In clinical trials with patients with advanced non-small cell lung carcinoma who progressed after chemotherapy, there was an objective response rate of 5% with a progression-free survival of 2.8 months and an overall survival of 9.5 months. As well, phase I/II studies showed positive dacomitinib activity despite prior failure with tyrosine kinase inhibitors. Phase III clinical trials (ARCHER 1050), done in patients suffering from advanced or metastatic non-small cell lung carcinoma with EGFR-activating mutations, reported a significant improvement in progression-free survival when compared with gefitinib.

C24H25CLFN5O2

469.9390

469.17

C, 61.34; H, 5.36; Cl, 7.54; F, 4.04; N, 14.90; O, 6.81

1110813-31-4

Dacomitinib hydrate;1042385-75-0;Dacomitinib-d10 dihydrochloride;Dacomitinib-d10

11511120

White to off-white solid powder

184-187 ºC

4.4

2

7

7

33

665

0

COC1=C(C=C2C(=C1)N=CN=C2NC3=CC(=C(C=C3)F)Cl)NC(=O)/C=C/CN4CCCCC4

LVXJQMNHJWSHET-AATRIKPKSA-N

InChI=1S/C24H25ClFN5O2/c1-33-22-14-20-17(24(28-15-27-20)29-16-7-8-19(26)18(25)12-16)13-21(22)30-23(32)6-5-11-31-9-3-2-4-10-31/h5-8,12-15H,2-4,9-11H2,1H3,(H,30,32)(H,27,28,29)/b6-5+

(E)-N-[4-(3-chloro-4-fluoroanilino)-7-methoxyquinazolin-6-yl]-4-piperidin-1-ylbut-2-enamide

Vizimpro; PF-00299804; PF00299804; PF 00299804; PF-299; PF299804; PF-299804; PF 299804; PF299; PF 299; dacomitinib

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.32 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 25.0 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.

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Solubility in Formulation 2: 2.5 mg/mL (5.32 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 25.0 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly..

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Solubility in Formulation 4: 1% DMSO+30% polyethylene glycol+1% Tween 80, pH 9: 10mg/mL

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