The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin inhibitor (mTOR) pathway is often constitutively activated in human tumor cells, providing unique opportunities for anticancer therapeutic intervention. NVP-BEZ235 is an imidazo [4,5-c]quinoline derivative that inhibits PI3K and mTOR kinase activity by binding to the ATP-binding cleft of these enzymes. In cellular settings using human tumor cell lines, this molecule is able to effectively and specifically block the dysfunctional activation of the PI3K pathway, inducing G 1 arrest. The cellular activity of NVP-BEZ235 translates well in in vivo models of human cancer. Thus, the compound was well tolerated, displayed disease stasis when administered orally, and enhanced the efficacy of other anticancer agents when used in in vivo combination studies. Ex vivo pharmacokinetic/pharmacodynamic analyses of tumor tissues showed a time-dependent correlation between compound concentration and PI3K/Akt pathway inhibition. Collectively, the preclinical data show that NVP-BEZ235 is a potent dual PI3K/mTOR modulator with favorable pharmaceutical properties. NVP-BEZ235 is currently in phase I clinical trials.

IGF-IR-mediated signaling promotes survival, anchorage-independent growth, and oncogenic transformation, as well as tumor growth and metastasis formation in vivo. NVP-AEW541 is a pyrrolo[2,3-d]pyrimidine derivative small molecular weight kinase inhibitor of the IGF-IR, capable of distinguishing between the IGF-IR (IC50 = 0.086 microM) and the closely related InsR (IC50 = 2.3 microM) in cells. As expected for a specific IGF-IR kinase inhibitor, NVP-AEW541 abrogates IGF-I-mediated survival and colony formation in soft agar at concentrations that are consistent with inhibition of IGF-IR autophosphorylation. In vivo, this orally bioavailable compound inhibits IGF-IR signaling in tumor xenografts and significantly reduces the growth of IGF-IR-driven fibrosarcomas. Thus, NVP-AEW541 represents a class of selective, small molecule IGF-IR kinase inhibitors with proven in vivo antitumor activity and potential therapeutic application.

A novel series of N-aryl-N'-pyrimidin-4-yl ureas has been optimized to afford potent and selective inhibitors of the fibroblast growth factor receptor tyrosine kinases 1, 2, and 3 by rationally designing the substitution pattern of the aryl ring. On the basis of its in vitro profile, compound 1h (NVP-BGJ398) was selected for in vivo evaluation and showed significant antitumor activity in RT112 bladder cancer xenografts models overexpressing wild-type FGFR3. These results support the potential therapeutic use of 1h as a new anticancer agent.

Introduction Heat shock protein 90 (HSP90) is a key component of a multichaperone complex involved in the posttranslational folding of a large number of client proteins, many of which play essential roles in tumorigenesis. HSP90 has emerged in recent years as a promising new target for anticancer therapies.

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.

Assessment of tumour vascularity may characterize malignancy as well as predict responsiveness to anti-angiogenic therapy. Non-invasive measurement of tumour perfusion and blood vessel permeability assessed as the transfer constant, K(trans), can be provided by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Using the orthotopic murine tumour model B16/BL6 melanoma, the small contrast agent GdDOTA (DOTAREM(R); Guerbet, Paris) was applied to assess the vascular transfer constant, K(trans), and interstitial leakage space, whereas intravascular iron oxide nanoparticles (Endorem(R); Guerbet, Paris) were used to detect relative tumour blood volume (rTBV), and in one experiment blood flow index (BFI). No correlations were observed between these four parameters (r(2) always <0.05). The B16/BL6 primary tumour and lymph-node cervical (neck) metastases produced high levels of the permeability/growth factor, VEGF. To probe the model, the novel VEGF receptor (VEGF-R) tyrosine kinase inhibitor, PTK787/ZK222584 (PTK/ZK) was tested for anti-tumour efficacy and its effects on DCE-MRI measured parameters of tumour vascularity. Data from the non-invasive measure of tumour vascularity were compared with a histological measurement of vasculature using the DNA-staining dye H33342. PTK/ZK inhibited growth of the primary and, particularly, cervical tumour metastases following chronic treatment for 2 weeks (50 or 100 mg/kg daily) of 1-week-old tumours, or with 1 week of treatment against more established (2-week-old) tumours. After chronic treatment with PTK/ZK, DCE-MRI detected significant decreases in K(trans) and interstitial leakage space, but not rTBV of both primary tumours and cervical metastases. Histological data at this time-point showed a significant decrease in blood vessel density of the cervical metastases but not the primary tumours. However, in the cervical metastases, the mean blood vessel width was increased by 38%, suggesting overall no marked change in blood volume. After acute (2-4 day) treatment, DCE-MRI of the cervical metastases demonstrated a significant decrease in K(trans) and interstitial leakage space and also in the initial area under the enhancement curve for GdDOTA (IAUC), but no change in the rTBV or BFI. Thus, significant changes could be detected in the DCE-MRI measurement of tumour uptake of a small contrast agent prior to changes in tumour size, which suggests that DCE-MRI could be applied in the clinic as a rapid and sensitive biomarker for the effects of VEGF-R inhibition on tumour blood vessel permeability and thus may provide an early marker for eventual tumour response.

PTK/ZK is a novel, oral angiogenesis inhibitor that specifically targets all 3 vascular endothelial growth factor (VEGF) receptor tyrosine kinases and is currently in phase III clinical trials. In early clinical trials, PTK/ZK demonstrated a dose-dependent reduction in tumor vascular parameters as measured by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and an acute increase in plasma VEGF levels. The reduction in tumor vascularity was significantly correlated with improved clinical outcome in patients with advanced colorectal cancer and liver metastases. To assess the predictive value of a mouse model of tumor metastases, comparisons were performed for the biological activity of PTK/ZK in the mouse model and in patients with liver metastases in the clinical phase I trials. An orthotopic, syngeneic mouse model was used: C57BL/6 mice injected in the ear with murine B16/BL6 melanoma cells which metastases to the cervical lymph-nodes. The primary tumor and spontaneous metastases express VEGF and VEGF receptors and respond to treatment with VEGFR tyrosine kinase inhibitors. PTK/ZK was administered orally, with assessments by DCE-MRI of the metastases and plasma VEGF taken predose and at 3 days posttreatment and efficacy determined at 7 days posttreatment. Dose-ranging studies in naive mice provided preclinical pharmacokinetic data, while two dose-escalation phase I studies provided clinical pharmacokinetic data. An exposure-response relationship was observed both for mouse metastases (measured as % tumor weight treated/control) and for human liver metastases (measured as % regression). In the B16/BL6 model, the active dose of 50 mg/kg PTK/ZK yielded 62.4 (+/- 16.0) h microM plasma exposure, which is comparable to the plasma area under the concentration time curve (AUC) achieved by the 1000 mg dose of PTK/ZK used in clinical trials. At this exposure level in clinical trials, DCE-MRI showed a reduction in the area under the enhancement curve (IAUC) to 47% of baseline. At a similar exposure in the PTK/ZK-treated mice, a reduction in IAUC to 75% of baseline was observed. Furthermore, at doses of 50 mg/kg PTK/ZK and above, an increase in plasma VEGF level 10 h after drug administration was observed in mice which was consistent with findings from the clinical trials. In conclusion, the preclinical pharmacodynamics of PTK/ZK correlate well with clinical activity in phase I trials over comparable exposures to the drug. Thus, data from this preclinical model proved to be consistent with and thus predictive of the biologic effects of PTK/ZK in phase I/II clinical trials.

Heat shock protein 90 (Hsp90) is a ubiquitously expressed molecular chaperone with ATPase activity involved in the conformational maturation and stability of key signaling molecules involved in cell proliferation, survival, and transformation. Through its ability to modulate multiple pathways involved in oncogenesis, Hsp90 has generated considerable interest as a therapeutic target. NVP-BEP800 is a novel, fully synthetic, orally bioavailable inhibitor that binds to the NH 2 -terminal ATP-binding pocket of Hsp90. NVP-BEP800 showed activity against a panel of human tumor cell lines and primary human xenografts in vitro at nanomolar concentrations. In A375 melanoma and BT-474 breast cancer cell lines, NVP-BEP800 induced client protein degradation (including ErbB2, B-Raf V600E , Raf-1, and Akt) and Hsp70 induction. Oral administration of NVP-BEP800 was well tolerated and induced robust antitumor responses in tumor xenograft models, including regression in the BT-474 breast cancer model. In these tumor models, NVP-BEP800 modulated Hsp90 client proteins and downstream signaling pathways at doses causing antitumor activity. NVP-BEP800 showed in vivo activity in a variety of dosing regimens covering daily to weekly schedules, potentially providing a high degree of flexibility in dose and schedule within the clinical setting. Overall, given the mechanism of action, preclinical activity profile, tolerability, and pharmaceutical properties, NVP-BEP800 is an exciting new oral Hsp90 inhibitor warranting further development.