Canertinib (CI-1033)

CI-1033 shows excellent potency for irreversible inhibition of erbB2 autophosphorylation in MDA-MB 453 cells. CI-1033 also shows high permeability in Caco-2 cells. ^[1] CI-1033 alone, significantly suppresses constitutively activated Akt and MAP kinase. CI-1033 in combination inhibits Akt and prevents increased levels of MAPK phosphorylation. CI-1033 stimulates p27 expression and p38 phosphorylation in MDA-MB-453 cells. ^[2] CI-1033 is highly specific to the erbB receptor family and not sensitive to PGFR, FGFR or IR even at 50 μM. CI-1033 shows high levels of inhibition in A431 cells expressing EGFR with IC50 of 7.4 nM. CI-1033 suppresses heregulin-stimulated tyrosine phosphorylation of erbB2, erbB3 and erbB4 with IC50 of 5, 14 and 10 nM, respectively. CI-1033 also inhibits expression of pp62^c-fos in response to heregulin. ^[3] CI-1033 is predicted to modify Cys773 covalently within the ATP binding site of the HER2 kinase and enhances destruction of both mature and immature ErbB-2 molecules. ^[4] CI-1033 induces a significant decrease in measurable phosphorylation of tyrosine residues 845 and 1068 of EGFR, which are responsible for Src and Ras/MAPK signaling respectively. The corresponding residues of Her-2, tyrosine residues 877 and 1248 are dephosphorylated significantly by CI-1033 at a concentration of 3 μM or higher. CI could block EGFR internalization and increase the rate of apoptosis in primary osteosarcoma cells in a titratable fashion. ^[5] In addition, CI-1033 inhibits the proliferation of TT, TE2, TE6 and TE10 cells significantly at 0.1 nM. ^[6]

CI-1033 shows impressive activity against A431 xenografts in nude mice at 5 mg/kg of body weight. ^[1] CI-1033 (20 to 80 mg/kg/d) achieves a high degree of tumor regressions in H125 xenograft models. ^[3] Oral administration of CI-1033 causes a marked inhibition of growth in TT, TE6 and TE10 xenografts in nude mice, without animal death and <10% weight loss. ^[6]

• Tyrosine Kinase Assays

  Enzyme assays for determination of IC50 are performed in 96-well filter plates in a total volume of 0.1 mL, containing 20 mM Hepes, pH 7.4, 50 mM sodium vanadate, 10 μM the ATP containing 0.5 mCi of [³²P]ATP, 20 mg of polyglutamic acid/tyrosine, 10 ng of EGFR tyrosine kinase, and appropriate dilutions of CI-1033. All components except the ATP are added to the well and the plate is incubated with shaking for 10 min at 25 °C. The reaction is started by adding [³²P]ATP, and the plate is incubated at 25 °C for another 10 min. The reaction is terminated by addition of 0.1 mL of 20% trichloroacetic acid (TCA). The plate is kept at 4 °C for at least 15 min to allow the substrate to precipitate. The wells are then washed five times with 0.2 mL of 10% TCA and 32P incorporation determined with a Wallac β plate counter.

• Cell lines

  TT, TE2, TE6 and TE10 cells

• Concentrations

  0.1-5.0 nM

• Incubation Time

  1, 3, 5 and 7 days

• Method

  Cells (1 × 10⁴) are seeded in each well of a 24-well plastic culture plate and left overnight in DMEM or RPMI-1640 supplemented with 10% FBS. The next morning, the cells are treated with the indicated concentrations of CI-1033 (0.1-5.0 nM) for varying periods (1, 3, 5 and 7 days). After treatment, the cells are counted using a Coulter counter. The percent of cell proliferation is calculated by this formula: treatment cell number/control cell number × 100 for each time period.

• Animal Models

  A431 xenografts established in nude mice

• Dosages

  ~18 mg/kg

• Administration

  Administered orally