CP-547632

Biological Activity

CP-547632 is a novel, potent vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor for cancer therapy. Signaling through vascular endothelial growth factor (VEGF) receptors (VEGFRs) is a key pathway initiating endothelial cell proliferation and migration resulting in angiogenesis, a requirement for human tumor growth and metastasis. CP-547632 was identified as a potent inhibitor of the VEGFR-2 and basic fibroblast growth factor (FGF) kinases (IC(50) = 11 and 9 nM, respectively). It is selective relative to epidermal growth factor receptor, platelet-derived growth factor beta, and other related TKs. CP-547632 also inhibits VEGF-stimulated autophosphorylation of VEGFR-2 in a whole cell assay with an IC(50) value of 6 nM. After oral administration of CP-547632 to mice bearing NIH3T3/H-ras tumors, VEGFR-2 phosphorylation in tumors was inhibited in a dose-dependent fashion (EC(50) = 590 ng/ml). CP-547632 did not significantly affect the pharmacologic profiles of paclitaxel and carboplatin. CP-547632 potently inhibits both basic FGF and VEGF-induced angiogenesis in vivo. CP-547632 is a well-tolerated, orally-bioavailable inhibitor presently under clinical investigation for the treatment of human malignancies.

Protocol

Cell Experiment
Cell lines	Porcine aortic endothelial cells
Preparation method	Cells were seeded at 1.6 × 105 cells/ml in 2-ml growth medium (Ham’s F-12 medium supplemented with 10% FBS, 50,000 units each penicillin and streptomycin, and 500 μg/ml gentamicin) per well in six-well plates. On day 2, the growth medium was replaced with serum-depleted medium (as above, but with 0.1% FBS and 0.1% BSA), and cells were incubated overnight. Immediately before compound addition, the medium was replaced with serum-depleted medium without BSA. Compounds were diluted in 100% DMSO, added to the cells at a final DMSO concentration of 0.25% v/v, and incubated at 37°C for 1 h. The cells were then stimulated with 500 ng/ml VEGF (Becton Dickinson, prepared in serum-depleted medium supplemented with 10 mm NaVO4) and incubated as above for an additional 8 min. The medium was removed and the cells washed once with PBS supplemented with 1 mm NaVO4, then lysed with 1 ml of immunoprecipitation assay buffer [10 mm Tris-HCl (pH 7.5), 50 mm NaCl, 25 mm EDTA, 1% NP40, 0.25% sodium deoxycholate, 2 mm NaVO4, and 1 EDTA-free complete protease inhibitor tablet per 25 ml]. Cell lysates were centrifuged at 14,000 rpm to pellet cellular debris, transferred to a new tube containing 4 μg anti-Flk-1 (Santa Cruz Biotechnology Laboratories; C20), and incubated with agitation overnight at 4°C. The antibody-protein complex was captured with protein A agarose beads (Santa Cruz Biotechnology Laboratories) for 30 min at 4°C and the protein boiled off in the presence of DTT. After electrophoresis and transfer to Immobilon-P membranes, the blots were probed with antibodies recognizing either the protein (monoclonal anti-Flk-1; Santa Cruz Biotechnology Laboratories; A3) or anti-PY-HRP. After incubation of the blot in enhanced chemiluminescence reagent (Amersham), bands were visualized on film or using the Lumi-ImagerF1 (Roche).
Concentrations	0~10 μ M
Incubation time	1 h

Animal Experiment
Animal models	athymic mice bearing Colo-205, DLD-1; and MDA-MB-231 cells tumour xenograft
Formulation	5% Gelucire
Dosages	6.25–100 mg/kg qd. for 24 days
Administration	oral gavage

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Chemical Information

References

A phase I/randomized phase II, non-comparative, multicenter, open label trial of CP-547,632 in combination with paclitaxel and carboplatin or paclitaxel and carboplatin alone as first-line treatment for advanced non-small cell lung cancer (NSCLC).
Cohen RB, et al. Cancer Chemother Pharmacol. 2007 Jun;60(1):81-9. PMID: 17031646.

Pharmacological characterization of CP-547,632, a novel vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor for cancer therapy.
Beebe JS, et al. Cancer Res. 2003 Nov 1;63(21):7301-9. PMID: 14612527.