Prioritization of EGFR/IGF-IR/VEGFR2 combination targeted therapies utilizing cancer models.

BACKGROUND: Rational strategies utilizing anticancer efficacy and biological principles are needed for the prioritization of specific combination targeted therapy approaches for clinical development, from among the many with experimental support. MATERIALS AND METHODS: Antibodies targeting epidermal growth factor receptor (EGFR) (cetuximab), insulin-like growth factor-1 receptor (IGF-IR) (IMC-A12) or vascular endothelial growth factor receptor 2 (VEGFR2) (DC101), were dosed alone or in combination, in 11 human tumor xenograft models established in mice. Efficacy readouts included the tumor burden and incidence of metastasis, as well as tumor active hypoxia inducible factor-1 (HIF-1), human VEGF and blood vessel density. RESULTS: Cetuximab and DC101 contributed potent and non-overlapping benefits to the combination approach. Moreover, DC101 prevented escape from IMC-A12 + cetuximab in a colorectal cancer model and cetuximab prevented escape from DC101 therapy in a pancreatic cancer model. CONCLUSION: Targeting VEGFR2 + EGFR was prioritized over other treatment strategies utilizing EGFR, IGF-IR and VEGFR2 antibodies. The criteria that proved to be valuable were a non-overlapping spectrum of anticancer activity and the prevention of resistance to another therapy in the combination.

Tumors established with cell lines selected for oxaliplatin resistance respond to oxaliplatin if combined with cetuximab.

PURPOSE: To establish whether cetuximab, a chimeric IgG1 antibody targeting epidermal growth factor receptor, has the potential to restore responsiveness to oxaliplatin in preclinical cancer models, as has been shown with irinotecan in irinotecan refractory metastatic colorectal cancer patients. EXPERIMENTAL DESIGN: The effects of cetuximab and oxaliplatin, alone or in combination, were tested in vitro and in vivo using human colorectal cancer cell lines selected for oxaliplatin resistance, as well as parental control cell lines. Evaluations were made of subcutaneous xenograft tumor growth in nu/nu athymic mice, as well as activation of mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) and AKT, expression of DNA repair genes, density of apurinic/apyrimidinic DNA damage, and accumulation of platinum-DNA adducts in vitro. RESULTS: Oxaliplatin + cetuximab efficacy in murine subcutaneous xenograft models was greater than that of monotherapies and independent of the responsiveness to oxaliplatin monotherapy. In vitro, cetuximab reduced expression of excision repair cross-complementation group 1 and XPF, which are key components of the nucleotide excision repair pathway involved in the excision of platinum-DNA adducts. In addition, cetuximab reduced expression of XRCC1, a component of the base excision repair pathway responsible for the repair of apurinic/apyrimidinic sites. Effects of cetuximab on DNA repair protein levels were downstream to effects on mitogen-activated protein kinase and AKT pathway activation. In line with effects on DNA repair protein expression, cetuximab increased the accumulation of platinum and apurinic/apyrimidinic sites on DNA during oxaliplatin treatment. CONCLUSIONS: Cetuximab has the potential to salvage the benefits of oxaliplatin in oxaliplatin-resistant colorectal cancer patients by reducing DNA repair capacity.

Synergistic antitumor effects of combined epidermal growth factor receptor and vascular endothelial growth factor receptor-2 targeted therapy.

PURPOSE: Combination therapies that target the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) pathways, are being actively tested for the treatment of cancer. In evaluating combination strategies, the ideal combination would be one in which the treatments interact in a way that is synergistic with regard to antitumor effects. Here, we have evaluated the interaction between anti-EGFR antibody Erbitux (cetuximab) and anti-VEGFR2 antibody, DC101, in preclinical models of pancreatic (BxPC-3) and colon (GEO) cancer. EXPERIMENTAL DESIGN: Analysis of the interaction between cetuximab and DC101 in vivo used a novel method for establishing the upper 95% confidence limits for the combination index (CI) of isobologram analyses, where CI < 1 indicates synergy. Assessment of tumor cell proliferation, apoptosis, VEGF production, and hypoxia, as well as tumor vascularization, was performed to gain insights into the mechanistic basis for synergy between agents targeting different tumor compartments. RESULTS: Monotherapy ED(50) values for tumor growth inhibition ranged from 1.8 to 2.3 mg/kg and 10.5 to 16.6 mg/kg for cetuximab and DC101, respectively. From the dose response of the combination treatment, it was determined that cetuximab and DC101 are synergistic in the BxPC-3 (CI = 0.1, P < 0.01) and GEO (CI = 0.1, P < 0.01) models. Overlapping effects on the tumor cell and vascular compartments form a basis for the interaction, with VEGF production and hypoxia-inducible factor 1alpha potentially acting as molecular links between EGFR and VEGFR2 inhibition. CONCLUSIONS: Results show antitumor synergy for combined EGFR and VEGFR2 targeted therapy, supporting the significant therapeutic potential of this combination strategy.

Targeting the platelet-derived growth factor receptor alpha with a neutralizing human monoclonal antibody inhibits the growth of tumor xenografts: implications as a potential therapeutic target.

Platelet-derived growth factor receptor alpha (PDGFRalpha) is a type III receptor tyrosine kinase that is expressed on a variety of tumor types. A neutralizing monoclonal antibody to human PDGFRalpha, which did not cross-react with the beta form of the receptor, was generated. The fully human antibody, termed 3G3, has a Kd of 40 pmol/L and blocks both PDGF-AA and PDGF-BB ligands from binding to PDGFRalpha. In addition to blocking ligand-induced cell mitogenesis and receptor autophosphorylation, 3G3 inhibited phosphorylation of the downstream signaling molecules Akt and mitogen-activated protein kinase. This inhibition was seen in both transfected and tumor cell lines expressing PDGFRalpha. The in vivo antitumor activity of 3G3 was tested in human glioblastoma (U118) and leiomyosarcoma (SKLMS-1) xenograft tumor models in athymic nude mice. Antibody 3G3 significantly inhibited the growth of U118 (P=0.0004) and SKLMS-1 (P <0.0001) tumors relative to control. These data suggest that 3G3 may be useful for the treatment of tumors that express PDGFRalpha.

Andrographolide, a potential cancer therapeutic agent isolated from Andrographis paniculata.

Andrographis paniculata plant extract is known to possess a variety of pharmacological activities. Andrographolide, the major constituent of the extract is implicated towards its pharmacological activity. We studied the cellular processes and targets modulated by andrographolide treatment in human cancer and immune cells. Andrographolide treatment inhibited the in vitro proliferation of different tumor cell lines, representing various types of cancers. The compound exerts direct anticancer activity on cancer cells by cell-cycle arrest at G0/G1 phase through induction of cell-cycle inhibitory protein p27 and decreased expression of cyclin-dependent kinase 4 (CDK4). Immunostimulatory activity of andrographolide is evidenced by increased proliferation of lymphocytes and production of interleukin-2. Andrographolide also enhanced the tumor necrosis factor-alpha production and CD marker expression, resulting in increased cytotoxic activity of lymphocytes against cancer cells, which may contribute for its indirect anticancer activity. The in vivo anticancer activity of the compound is further substantiated against B16F0 melanoma syngenic and HT-29 xenograft models. These results suggest that andrographolide is an interesting pharmacophore with anticancer and immunomodulatory activities and hence has the potential for being developed as a cancer therapeutic agent.

Preclinical rationale for combining an EGFR antibody with cisplatin/gemcitabine for the treatment of NSCLC.

BACKGROUND: Although the addition of epidermal growth factor receptor (EGFR) antibodies to various platinum-based chemotherapy regimens for non-small cell lung cancer (NSCLC) is being actively pursued in the clinic, rationale for the prioritization of specific regimens is lacking. MATERIALS AND METHODS: We evaluated the antitumor effects of necitumumab, a recombinant human IgG1 antibody targeting EGFR, in combination with cisplatin plus gemcitabine, pemetrexed, or paclitaxel in a panel of 9 subcutaneous tumor models of NSCLC established in nu/nu athymic mice. RESULTS: Necitumumab in combination with cisplatin/gemcitabine was particularly effective, although interestingly, the mechanisms underlying these benefits were model dependent. For example, increased tumor cell apoptosis contributed towards combination efficacy in the A549 model, in association with increased expression of hsa-miR-29b and reduced expression of antiapoptotic genes including DNA methyltransferase DNMT3B, commonly up-regulated in patients with NSCLC. Such inverse effects of combination therapy on DNMT3B and hsa-miR-29b expression were found in multiple models. Importantly, in the A549 model, hsa-miR-29b down-regulation of DMNT3b reduced promoter methylation of tumor suppressor genes such as Cell adhesion molecule 1 (CADM1), Ras associated (RalGDS/AF-6) domain family member 1 (RASSF1), and Fragile histidine triad gene (FHIT), increasing their expression. CONCLUSION: These results offer a preclinical rationale for combining an EGFR antibody with cisplatin/gemcitabine for patients with NSCLC, and provide potential molecular biomarkers for tailoring therapy.

Development of a fully human anti-PDGFRbeta antibody that suppresses growth of human tumor xenografts and enhances antitumor activity of an anti-VEGFR2 antibody.

Platelet-derived growth factor receptor beta (PDGFRbeta) is upregulated in most of solid tumors. It is expressed by pericytes/smooth muscle cells, fibroblast, macrophage, and certain tumor cells. Several PDGF receptor-related antagonists are being developed as potential antitumor agents and have demonstrated promising antitumor activity in both preclinical and clinical settings. Here, we produced a fully human neutralizing antibody, IMC-2C5, directed against PDGFRbeta from an antibody phage display library. IMC-2C5 binds to both human and mouse PDGFRbeta and blocks PDGF-B from binding to the receptor. IMC-2C5 also blocks ligand-stimulated activation of PDGFRbeta and downstream signaling molecules in tumor cells. In animal studies, IMC-2C5 significantly delayed the growth of OVCAR-8 and NCI-H460 human tumor xenografts in nude mice but failed to show antitumor activities in OVCAR-5 and Caki-1 xenografts. Our results indicate that the antitumor efficacy of IMC-2C5 is primarily due to its effects on tumor stroma, rather than on tumor cells directly. Combination of IMC-2C5 and DC101, an anti-mouse vascular endothelial growth factor receptor 2 antibody, resulted in significantly enhanced antitumor activity in BxPC-3, NCI-H460, and HCT-116 xenografts, compared with DC101 alone, and the trend of additive effects to DC101 treatment in several other tumor models. ELISA analysis of NCI-H460 tumor homogenates showed that IMC-2C5 attenuated protein level of vascular endothelial growth factor and basic fibroblast growth factor elevated by DC101 treatment. Finally, IMC-2C5 showed a trend of additive effects when combined with DC101/chemotherapy in MIA-PaCa-2 and NCI-H460 models. Taken together, these results lend great support to the use of PDGFRbeta antagonists in combination with other antiangiogenic agents in the treatment of a broad range of human cancers.

An antibody directed against PDGF receptor beta enhances the antitumor and the anti-angiogenic activities of an anti-VEGF receptor 2 antibody.

Platelet-derived growth factor (PDGF) and its receptors (PDGFR) play important roles in tumorigenesis through stimulating tumor growth and promoting angiogenesis via enhancing pericyte recruitment and vessel maturation. Here we produced a neutralizing antibody, 1B3, directed against mouse PDGFRbeta. 1B3 binds to PDGFRbeta with high affinity (9x10(-11)M) and blocks PDGF-BB from binding to the receptor with an IC(50) of approximately 1.2 nM. The antibody also blocks ligand-stimulated activation of PDGFRbeta and downstream signaling molecules, including Akt and MAPK p42/44, in tumor cells. In animal studies, 1B3 significantly enhanced the antitumor and the anti-angiogenic activities of DC101, an antibody directed against mouse vascular endothelial growth factor receptor 2, in a pancreatic (BxPC-3) and a non-small cell lung (NCI-H460) tumor xenograft models. Treatment with the combination of 1B3 and DC101 in BxPC-3 xenograft-bearing mice resulted in tumor regression in 58% of mice compared to that in 18% of mice treated with DC101 alone. Taken together, these results lend great support to use PDGFRbeta antagonists in combinations with other antitumor and/or anti-angiogenic agents in the treatment of a variety of cancers.

N-(4-{[4-(1H-Benzoimidazol-2-yl)-arylamino]-methyl}-phenyl)-benzamide derivatives as small molecule heparanase inhibitors.

A novel class of N-(4-{[4-(1H-benzoimidazol-2-yl)-arylamino]-methyl}-phenyl)-benzamides are described as inhibitors of the endo-beta-glucuronidase heparanase. Among them are N-(4-{[4-(1H-benzoimidazol-2-yl)-phenylamino]-methyl}-phenyl)-3-bromo-4-methoxy-benzamide (15h), and N-(4-{[5-(1H-benzoimidazol-2-yl)-pyridin-2-ylamino]-methyl}- phenyl)-3-bromo-4-methoxy-benzamide (23) which displayed good heparanase inhibitory activity (IC(50) 0.23-0.29 microM), with the latter showing oral exposure in mice.

Novel 6,7-diphenyl-2,3,8,8a-tetrahydro-1H-indolizin-5-one analogues as cytotoxic agents.

A series of 6,7-diphenyl-2,3,8,8a-tetrahydro-1H-indolizin-5-one analogues were synthesized and evaluated for cytotoxic activity against eight human cancer cell lines. Compounds 18, 21, 28, 29, 30 and 31 showed cytotoxic activity with GI(50) values in the range of 2.1-8.1 microM concentration. Among these, compounds 21 and 28 exhibited good pharmacokinetic properties. These compounds were further evaluated for their in vivo efficacy in modified hollow fibre assay (HFA).

Biological investigation and structure-activity relationship studies on azadirone from Azadirachta indica A. Juss.

Azadirone 1, a limonoidal constituent of Azadirachta indica is found to possess potent cytotoxic activity against a panel of human cancer cell lines in our in vitro studies. In vitro screening of a number of semi-synthetic analogues of 1 revealed that the alpha,beta-unsaturated enone moiety or its equivalent conjugated system in A-ring, C-7 acetyloxy/chloroacetyloxy or keto group in B-ring and the furan moiety are responsible for the activity of 1 and its analogues. Compound 1 and two of the semi-synthetic analogues 10 and 13 were found to possess good in vivo antitumor activity in modified hollow fiber animal models.

Novel indolo[2,1-b]quinazoline analogues as cytostatic agents: synthesis, biological evaluation and structure-activity relationship.

In our endeavor to design and synthesize novel anticancer agents, a new series of indoloquinazoline compounds were prepared and tested initially for anticancer activity in vitro against a panel of human cancer cell lines. Most of these compounds exhibited cytotoxic activity in in vitro screens. Compounds were selected and further evaluated using a modified Hollow Fiber Assay for their preliminary in vivo activity against 12 cell lines implanted in the subcutaneous and intraperitoneal compartments in mice. The results indicate that these compounds may constitute a new class of anticancer agents.