Ennio Cavalletti

E-3810 Is a Potent Dual Inhibitor of VEGFR and FGFR that Exerts Antitumor Activity in Multiple Preclinical Models

Tumor angiogenesis is a degenerate process regulated by a complex network of proangiogenic factors. Existing antiangiogenic drugs used in clinic are characterized by selectivity for specific factors. Antiangiogenic properties might be improved in drugs that target multiple factors and thereby address the inherent mechanistic degeneracy in angiogenesis. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) family members and their cognate receptors are key players in promoting tumor angiogenesis. Here we report the pharmacologic profile of E-3810, a novel dual inhibitor of the VEGF and FGF receptors. E-3810 potently and selectively inhibited VEGF receptor (VEGFR)-1, -2, and -3 and FGF receptor (FGFR)-1 and -2 kinases in the nanomolar range. Ligand-dependent phosphorylation of VEGFR-2 and FGFR-1 was suppressed along with human vascular endothelial cell growth at nanomolar concentrations. In contrast, E-3810 lacked cytotoxic effects on cancer cell lines under millimolar concentrations. In a variety of tumor xenograft models, including early- or late-stage subcutaneous and orthotopic models, E-3810 exhibited striking antitumor properties at well-tolerated oral doses administered daily. We found that E-3810 remained active in tumors rendered nonresponsive to the general kinase inhibitor sunitinib resulting from a previous cycle of sunitinib treatment. In Matrigel plug assays performed in nude mice, E-3810 inhibited basic FGF-induced angiogenesis and reduced blood vessel density as assessed by histologic analysis. Dynamic contrast-enhanced magnetic resonance imaging analysis confirmed that E-3810 reduced the distribution of angiogenesis-sensitive contrast agents after only 5 days of treatment. Taken together, our findings identify E-3810 as a potent antiangiogenic small molecule with a favorable pharmacokinetic profile and broad spectrum antitumor activity, providing a strong rationale for its clinical evaluation.

BBR 2778, an aza-anthracenedione endowed with preclinical anticancer activity and lack of delayed cardiotoxicity

With the aim to provide second-generation anthracenedione analogues endowed with reduced side effects and a wider spectrum of action than mitoxantrone and doxorubicin, a large number of new molecules bearing nitrogen atoms in the chromophore was synthesized and screened in vitro and in vivo. From this screening, BBR 2778 (6,9-bis[(2-aminoethyl)amino] benzo[g]isoquinoline-5,10-dione dimaleate) emerged as the most interesting compound. BBR 2778 was tested in vitro on several murine and human tumor cell lines and showed cytotoxic potency lower than that of mitoxantrone and doxorubicin. BBR 2778 was more cytotoxic in leukemia and lymphoma cell lines than in solid tumor cell lines. Although against in vivo models BBR 2778 was less potent than mitoxantrone and doxorubicin, its antitumor activity was equal or superior (in certain tumor models) to that of the above standard compounds. In particular, BBR 2778 was curative against L1210 murine leukemia and YC-8 murine lymphoma. Moreover, it showed an antitumor activity comparable to that of mitoxantrone and doxorubicin on solid tumors. No cardiotoxic effect of BBR 2778 in animals not pretreated with anthracyclines was observed compared to standards. In light of its spectrum of activity and marked efficacy against lymphomas and leukemias over a wide dose range, together with its lack of delayed cardiotoxicity, BBR 2778 has been entered in clinical studies.

The Tyrosine Kinase Inhibitor E-3810 Combined with Paclitaxel Inhibits the Growth of Advanced-Stage Triple-Negative Breast Cancer Xenografts

E-3810 is a novel small molecule that inhibits VEGF receptor-1, -2, and -3 and fibroblast growth factor receptor-1 tyrosine kinases at nmol/L concentrations currently in phase clinical II. In preclinical studies, it had a broad spectrum of antitumor activity when used as monotherapy in a variety of human xenografts. We here investigated the activity of E-3810 combined with different cytotoxic agents in a MDA-MB-231 triple-negative breast cancer xenograft model. The molecule could be safely administered with 5-fluorouracil, cisplatin, and paclitaxel. The E-3810–paclitaxel combination showed a striking activity with complete, lasting tumor regressions; the antitumor activity of the combination was also confirmed in another triple-negative breast xenograft, MX-1. The activity was superior to that of the combinations paclitaxel+brivanib and paclitaxel+sunitinib. Pharmacokinetics studies suggest that the extra antitumor activity of the combination is not due to higher paclitaxel tumor levels, which in fact were lower in mice pretreated with all three kinase inhibitors, and the paclitaxel plasma levels excluded reduced drug availability. Pharmacodynamic studies showed that E-3810, brivanib, and sunitinib given as single agents or in combination with paclitaxel reduced the number of vessels, but did not modify vessel maturation. Reduced tumor collagen IV and increased plasma collagen IV, associated with increased matrix metalloproteinases (MMP), particularly host MMP-9, indicate a proteolytic remodeling of the extracellular matrix caused by E-3810 that in conjunction with the cytotoxic effect of paclitaxel on the tumor cells (caspase-3/7 activity) may contribute to the striking activity of their combination. These data support the therapeutic potential of combining E-3810 with conventional chemotherapy. Mol Cancer Ther; 12(2); 131–40. ©2012 AACR.

ATP Non-Competitive Ser/Thr Kinase Inhibitors as Potential Anticancer Agents

Protein kinases are one of the largest known families of enzyme characterized by having a well conserved ATP binding pocket. Most of the synthetic kinase inhibitors are ATP-competitive, but display some potential problems, like selectivity, discrepancy between the in vitro and in vivo inhibition assays and an high risk of developing mutation inside the ATP-binding pocket. Recently some new inhibitors with a non-competitive mechanism of action were reported, with interesting results both in vitro and in vivo.

Abstract 1367: The dual selective VEGFR-FGFR kinases inhibitor E-3810 decreases tumor perfusion and inhibits tumor growth: an analysis using dynamic contrast-enhanced magnetic resonance imaging

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: E-3810 is a potent VEGFR1, 2 and 3 and FGFR 1 kinases inhibitor showing potent anti-angiogenic and antitumor activity in several in vitro and in vivo mouse models (Proceedings AACR-EORTC-NCI Meeting, Boston, November 2009; Abstracts No. 252 and 257). The aim of this study was to investigate, in a rat model, the antitumor activity of E-3810 and its anti-angiogenic effects by using DCE-MRI and to correlate the response with a panel of biological markers. Methods: Nude rats were subcutaneously xenografted with human Calu-6 lung cancer cells and treated orally once daily for 14 consecutive days, either with E-3810 − 10 mpk (dose chosen on the basis of a preliminary study in the same model) or Sorafenib − 100 mpk. Tumor growth and parameters reflecting tumor vascular permeability/perfusion (Ktrans) were evaluated by T2-weighted MRI and by T1-weighted DCE-MRI using Gd-DTPA (Magnevist®) as contrast agent at D-1, D1, D3, D7 and D14. The number, maturity and functionality of tumor vessels were assessed using CD31/α-SMA colocalisation by IHC and injection of Hoechst fluorescent dye on D1, D3, D7 and D14. On the same days, the level of circulating endothelial cells (CEC) and collagen IV were measured by FACS and ELISA assays. Results: In comparison to vehicle-treated animals, a marked tumor growth inhibition was observed in E-3810-treated rats (max T/C 21%) with no remarkable signs of toxicity. In the same animals, Ktrans value in the tumor rim was significantly decreased (up to 49% on D14). This DCE-MRI finding was supported by the significant decrease in the Hoechst stained area observed at D1 (−97%), D7 (−96%) and D14 (−75%). In addition, the number of tumor vessels (both CD31 and colocalized CD31/α-SMA) was significantly decreased at D3 (−72%), D7 (−68%) and D14 (−86%) in E-3810-treated rats. Always in comparison to vehicle group, no significant changes in CEC were observed after treatment with E-3810, whereas collagen IV levels significantly increased at D14 (+38). Similar effects on all the above mentioned parameters were observed in Sorafenib treated animals. Conclusions: E-3810 displayed a marked antitumor activity in the model of Calu-6 tumor bearing nude rats. The antitumor effect is coupled with a strong antiangiogenic activity, as indicated by decrease in vascular permeability/perfusion and decrease of microvessels density that occur early on and are maintained throughout the treatment period. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1367.

Abstract C192: Molecular mechanisms of antitumor activity of the combination of E-3810 and paclitaxel in MDA-MB-231 triple-negative breast xenograft.

E-3810 is a small molecule inhibitor of VEGFR-1, -2 and -3 and FGFR-1 tyrosine kinases with IC50 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C192.

Abstract 2574: Striking activity of E-3810 combined with paclitaxel and 5FU in the triple negative breast cancer model MDA-MB-231

E-3810 is a novel small molecule that selectively inhibits VEGFR-1, -2 and -3 and FGFR-1 tyrosine kinases with IC50 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2574. doi:10.1158/1538-7445.AM2011-2574

Abstract A262: Antiangiogenic proprieties of E‐3810, a new dual inhibitor of VEGF and FGF receptors

E‐3810, 6‐[[7‐[(1‐aminocyclopropyl)methoxy]‐6‐methoxy‐4‐quinolyl]oxy]‐N‐methyl‐naphthalene‐1‐carboxamide, is a novel small molecule that potently and selectively inhibits VEGFR1‐3 and FGFR‐1 tyrosine kinases. E‐3810 has shown antitumor activity against various types of cancer xenografts with good tolerability. The anti‐angiogenic proprieties of E‐3810 were investigated in vitro on endothelial cells and in vivo in angiogenesis‐induced models and human tumor xenografts. E‐3810 inhibited VEGF‐ and FGF‐2‐induced HUVEC (human umbilical vein endothelial cells) proliferation with an IC50 of 40–50 nM. The ability of E‐3810 to inhibit angiogenesis in vivo was assessed in the Matrigel plug assay, where angiogenesis is induced by FGF‐2 embedded in Matrigel pellets, implanted subcutaneously in mice. Treatment with E‐3810 (20 mg/kg p.o., daily for 6 days) completely inhibited the angiogenic response measured by hemoglobin content. The effect on tumor angiogenesis was investigated in the human A498 renal carcinoma xenografted subcutaneously in nude mice. Oral treatment with E‐3810 for up 30 days at the optimal antitumor dose of 20 mg/kg led to significant tumor growth delay (Treated ‐Controls = 24 days). E‐3810 significantly decreased blood vessel density and increased vessel maturation index (assessed by immunostaining with anti CD31 and CD31/ SMA antibodies); this was accompanied by decreased expression of stroma collagen IV and increased tumor necrosis. A498 xenografts growing in nude mice were also analyzed by DCE‐MRI (dynamic contrast‐enhanced magnetic resonance imaging) before and after 5 and 12 days of oral treatments with 20 mg/kg E‐ 3810. The kinetic analysis of the distribution of the contrast agent (Gadocoletic Acid Trisodium Salt, B22956/1, Bracco S.p.a., Italy) showed significant reduction in perfusion, calculated applying the IAUC method over the first 30 minutes post contrast injection, in E‐3810 treated tumors compared with both baseline values and untreated controls already after 5 days of treatment. These results show a significant antiangiogenic activity of E‐3810, which is likely to contribute to its potent antitumor activity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A262.

Abstract A257: Antitumor activity of E‐3810, a new, potent and selective dual inhibitor of VEGF and FGF receptors

E‐3810, 6‐[[7‐[(1‐aminocyclopropyl)methoxy]‐6‐methoxy‐4‐quinolyl]oxy]‐N‐methyl‐naphthalene‐1‐carboxamide, is a novel small molecule that potently and selectively inhibits VEGFR1‐3 and FGFR‐1 tyrosine kinases (TKs) with IC50 in the 10‐20 nM range. Concentrations at least 1‐order higher were required to inhibit FGFR‐2 and 3, PDGFRα and β and c‐Kit receptors and no relevant activity was seen against different cancer‐related TKs. E‐3810 1‐10 nM inhibited the ligand‐dependent phosphorylation of VEGFR‐2 and FGFR‐1 and the downstream phosphorylation of Erk in HUVEC cells; inhibition of PDGFR‐ auto‐phosphorylation in NIH‐3T3 cells was detected at concentrations higher than 100 nM. E‐3810 inhibited the VEGF‐ and FGF‐driven growth of HUVEC cells (IC50 40‐50 nM), while anti‐proliferative activity on different human cancer cell lines was shown at 10‐30 M concentrations. E‐3810 was tested in different human tumor xenografts in nude mice, including colon HT‐29, ovarian A‐2780, renal A498, RXF393 and SKN12I carcinomas. A dose dependent antitumor effect was observed in a range from 10 to 40 mg/kg given orally for 30 consecutive days. The optimal dose schedule, 20 mg/kg for 30 days, was well tolerated and caused up to 90% suppression of tumor growth. Sustained growth inhibition was also seen when treatment was repeated after a 2‐week dosing interval or when dosing was initiated at a larger tumor volume (average 400 mm3). Tumor regression was observed in the RXF393 xenograft. E‐3810 showed antitumor activity equal or better than Sunitinib and Brivanib at their respective optimal doses. Pharmacokinetic studies in mice demonstrated high oral bioavailability, high volume of distribution and terminal half life of 4 hrs. After single and repeated administration E‐3810 systemic exposure was proportional to the dose, without any accumulation after repeated doses. The good bioavailability and the remarkable antitumor activity of E‐3810 in several tumor xenografts make this compound an interesting candidate for clinical development. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A257.