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Aberrant epidermal growth factor receptor (EGFR) and ErbB2 expression are associated with advanced disease and poor patient prognosis in many tumor types (breast, lung, ovarian, prostate, glioma, gastric, and squamous carcinoma of head and neck). In addition, a constitutively active EGFR type III deletion mutant has been identified in non-small cell lung cancer, glioblastomas, and breast tumors. Hence, members of the EGFR family are viewed as promising therapeutic targets in the fight against cancer. In a similar vein, vascular endothelial growth factor (VEGF) receptor kinases are also promising targets in terms of an antiangiogenic treatment strategy. AEE788, obtained by optimization of the 7H-pyrrolo[2,3-d]pyrimidine lead scaffold, is a potent combined inhibitor of both epidermal growth factor (EGF) and VEGF receptor tyrosine kinase family members on the isolated enzyme level and in cellular systems. At the enzyme level, AEE788 inhibited EGFR and VEGF receptor tyrosine kinases in the nm range (IC(50)s: EGFR 2 nm, ErbB2 6 nm, KDR 77 nm, and Flt-1 59 nm). In cells, growth factor-induced EGFR and ErbB2 phosphorylation was also efficiently inhibited (IC(50)s: 11 and 220 nm, respectively). AEE788 demonstrated antiproliferative activity against a range of EGFR and ErbB2-overexpressing cell lines (including EGFRvIII-dependent lines) and inhibited the proliferation of epidermal growth factor- and VEGF-stimulated human umbilical vein endothelial cells. These properties, combined with a favorable pharmacokinetic profile, were associated with a potent antitumor activity in a number of animal models of cancer, including tumors that overexpress EGFR and or ErbB2. Oral administration of AEE788 to tumor-bearing mice resulted in high and persistent compound levels in tumor tissue. Moreover, AEE788 efficiently inhibited growth factor-induced EGFR and ErbB2 phosphorylation in tumors for >72 h, a phenomenon correlating with the antitumor efficacy of intermittent treatment schedules. Strikingly, AEE788 also inhibited VEGF-induced angiogenesis in a murine implant model. Antiangiogenic activity was also apparent by measurement of tumor vascular permeability and interstitial leakage space using dynamic contrast enhanced magnetic resonance imaging methodology. Taken together, these data indicate that AEE788 has potential as an anticancer agent targeting deregulated tumor cell proliferation as well as angiogenic parameters. Consequently, AEE788 is currently in Phase I clinical trials in oncology.

Antiangiogenic therapy is a promising new strategy of inhibiting tumor growthand formation of metastases. Recently, a number of compounds with different effects on tumor endothelial cells have entered clinical trials and revealed the need for diagnostic methods to detect their biological activity. Dynamic enhanced magnetic resonance imaging (dyMRI) is used in most clinical trials with antiangiogenic active compounds. We evaluated this method by using PTK787/ZK 222584, a specific inhibitor of the VEGF-receptor tyrosine kinases, which showed antitumoral and antiangiogenic activity in a murine renal cell carcinoma (RENCA) model. After intrarenal application of RENCA cells, mice developed a primary tumor and metastases to the lung and abdominal lymph nodes. After daily oral therapy for 21 days with either PTK787/ZK 222584 at a dose of 50 mg/kg or vehicle, primary tumors of all animals were analyzed by dyMRI. Gadolinium-DOTA (Dotarem) was used as a contrast agent to detect vessel permeability and contrast agent extravasation, whereas intravascular iron oxide nanoparticles (Endorem) were used to detect partial tumor blood volume. Additionally, vessel density, architecture, diameter, and blood flow velocity were investigated by appropriate methods. Surprisingly, no changes in extravasation occurred under treatment with PTK787/ZK 222584 as compared with the control group, whereas a significant decrease in vessel permeability occurred. Furthermore, an increase in partial blood volume was found in the PTK787/ZK 222584-treated group, although vessel density was reduced as seen by histology. Using the corrosion cast technique, reduction in vessel density was significant but not very pronounced and predominantly attributable to the loss of microvessels only. This finding correlated with a shift to large vessel diameters in the primary tumors of PTK787/ZK 222584-treated animals and with reduction of blood flow velocity in the tumor feeding renal artery. From these findings, we conclude that the treatment with PTK787/ZK 222584 primarily reduces the number of tumor microvessels, accompanied by a hemodynamic dilation of the remaining vessels. This dilation could influence the result of dyMRI such that no change in extravasation or even an increase in partial tumor blood volume could be observed.