Lauren Bazinet

An orally delivered small-molecule formulation with antiangiogenic and anticancer activity

Targeting angiogenesis, the formation of blood vessels, is an important modality for cancer therapy. TNP-470, a fumagillin analog, is among the most potent and broad-spectrum angiogenesis inhibitors. However, a major clinical limitation is its poor oral availability and short half-life, necessitating frequent, continuous parenteral administration. We have addressed these issues and report an oral formulation of TNP-470, named Lodamin. TNP-470 was conjugated to monomethoxy-polyethylene glycol–polylactic acid to form nanopolymeric micelles. This conjugate can be absorbed by the intestine and selectively accumulates in tumors. Lodamin significantly inhibits tumor growth, without causing neurological impairment in tumor-bearing mice. Using the oral route of administration, it first reaches the liver, making it especially efficient in preventing the development of liver metastasis in mice. We show that Lodamin is an oral nontoxic antiangiogenic drug that can be chronically administered for cancer therapy or metastasis prevention.

Endostatin lowers blood pressure via nitric oxide and prevents hypertension associated with VEGF inhibition

Antiangiogenesis therapy has become a vital part of the armamentarium against cancer. Hypertension is a dose-limiting toxicity for VEGF inhibitors. Thus, there is a pressing need to address the associated adverse events so these agents can be better used. The hypertension may be mediated by reduced NO bioavailability resulting from VEGF inhibition. We proposed that the hypertension may be prevented by coadministration with endostatin (ES), an endogenous angiogenesis inhibitor with antitumor effects shown to increase endothelial NO production in vitro. We determined that Fc-conjugated ES promoted NO production in endothelial and smooth muscle cells. ES also lowered blood pressure in normotensive mice and prevented hypertension induced by anti-VEGF antibodies. This effect was associated with higher circulating nitrate levels and was absent in eNOS-knockout mice, implicating a NO-mediated mechanism. Retrospective study of patients treated with ES in a clinical trial revealed a small but significant reduction in blood pressure, suggesting that the findings may translate to the clinic. Coadministration of ES with VEGF inhibitors may offer a unique strategy to prevent drug-related hypertension and enhance antiangiogenic tumor suppression.

Melanocyte-secreted fibromodulin promotes an angiogenic microenvironment

Studies have established that pigmentation can provide strong, protective effects against certain human diseases. For example, angiogenesis-dependent diseases such as wet age-related macular degeneration and infantile hemangioma are more common in light-skinned individuals of mixed European descent than in African-Americans. Here we found that melanocytes from light-skinned humans and albino mice secrete high levels of fibromodulin (FMOD), which we determined to be a potent angiogenic factor. FMOD treatment stimulated angiogenesis in numerous in vivo systems, including laser-induced choroidal neovascularization, growth factor-induced corneal neovascularization, wound healing, and Matrigel plug assays. Additionally, FMOD enhanced vascular sprouting during normal retinal development. Deletion of Fmod in albino mice resulted in a marked reduction in the amount of neovascularization induced by retinal vein occlusion, corneal growth factor pellets, and Matrigel plugs. Our data implicate the melanocyte-secreted factor FMOD as a key regulator of angiogenesis and suggest an underlying mechanism for epidemiological differences between light-skinned individuals of mixed European descent and African-Americans. Furthermore, inhibition of FMOD in humans has potential as a therapeutic strategy for treating angiogenesis-dependent diseases.

Broad spectrum antiangiogenic treatment for ocular neovascular diseases.

Pathological neovascularization is a hallmark of late stage neovascular (wet) age-related macular degeneration (AMD) and the leading cause of blindness in people over the age of 50 in the western world. The treatments focus on suppression of choroidal neovascularization (CNV), while current approved therapies are limited to inhibiting vascular endothelial growth factor (VEGF) exclusively. However, this treatment does not address the underlying cause of AMD, and the loss of VEGFs neuroprotective can be a potential side effect. Therapy which targets the key processes in AMD, the pathological neovascularization, vessel leakage and inflammation could bring a major shift in the approach to disease treatment and prevention. In this study we have demonstrated the efficacy of such broad spectrum antiangiogenic therapy on mouse model of AMD. Methods and Findings Lodamin, a polymeric formulation of TNP-470, is a potent broad-spectrum antiangiogenic drug. Lodamin significantly reduced key processes involved in AMD progression as demonstrated in mice and rats. Its suppressive effects on angiogenesis, vascular leakage and inflammation were studied in a wide array of assays including; a Matrigel, delayed-type hypersensitivity (DTH), Miles assay, laser-induced CNV and corneal micropocket assay. Lodamin significantly suppressed the secretion of various pro-inflammatory cytokines in the CNV lesion including monocyte chemotactic protein-1 (MCP-1/Ccl2). Importantly, Lodamin was found to regress established CNV lesions, unlike soluble fms-like tyrosine kinase-1 (sFlk-1). The drug was found to be safe in mice and have little toxicity as demonstrated by electroretinography (ERG) assessing retinal and by histology. Conclusions Lodamin, a polymer formulation of TNP-470, was identified as a first in its class, broad-spectrum antiangiogenic drug that can be administered orally or locally to treat corneal and retinal neovascularization. Several unique properties make Lodamin especially beneficial for ophthalmic use. Our results support the concept that broad spectrum antiangiogenic drugs are promising agents for AMD treatment and prevention.

Dendritic Cells Augment Choroidal Neovascularization

PURPOSE Dendritic cells (DCs) are innate immune cells that have recently been shown to support angiogenesis in tumors, endometriosis, and lymph nodes. A major cause of legal blindness is wet age-related macular degeneration (wet ARMD), wherein abnormal blood vessels grow under the retina, an abnormality also referred to as choroidal neovascularization (CNV). The purpose of the present study was to investigate the role of DCs in the development of CNV. METHODS Laser photocoagulation was used to induce CNV in C57BL/6J mice. The authors analyzed CNV lesions for the presence of DCs using flow cytometry and immunostaining at designated times. They also analyzed the effects of intravenous DC transplantation on CNV development by measuring the lesion area using confocal microscopy 1 week after laser injury. RESULTS The authors analyzed CNV lesions for the presence of DCs by flow cytometry and observed that CD11c(+) major histocompatibility complex (MHC) class II(+) DCs transiently infiltrated the CNV lesions, reaching a peak at 2 to 4 days after laser injury. These DCs were mostly immature (CD11c(+) MHCII(low)) and expressed vascular endothelial growth factor receptor 2. Immunostaining of laser-induced CNV lesions confirmed that DCs are located at the sites of newly formed blood vessels. Intravenously injected DCs incorporated into the CNV lesions. However, only immature DCs enhanced CNV size. CONCLUSIONS These results suggest a role for DCs in promoting angiogenesis and lesion growth in laser-induced CNV. The present data suggest that DCs may represent potential cellular targets for therapeutic intervention in wet ARMD.

1,2,3,4,6-Penta-O-galloyl-β-D-glucopyranose inhibits angiogenesis via inhibition of capillary morphogenesis gene 2.

Capillary morphogenesis gene 2 (CMG2) is a transmembrane extracellular matrix binding protein that is also an anthrax toxin receptor. We have shown that high-affinity CMG2 binders can inhibit angiogenesis and tumor growth. We recently described a high-throughput FRET assay to identify CMG2 inhibitors. We now report the serendipitous discovery that PGG (1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose) is a CMG2 inhibitor with antiangiogenic activity. PGG is a gallotannin produced by a variety of medicinal plants that exhibits a wide variety of antitumor and other activities. We find that PGG inhibits CMG2 with a submicromolar IC50 and it also inhibits the migration of human dermal microvascular endothelial cells at similar concentrations in vitro. Finally, oral or intraperitoneal administration of PGG inhibits angiogenesis in the mouse corneal micropocket assay in vivo. Together, these results suggest that a portion of the in vivo antitumor activity of PGG may be the result of antiangiogenic activity mediated by inhibition of CMG2.

Suppression of Autoimmune Retinal Inflammation by an Antiangiogenic Drug

Chronic and recurrent uveitis account for approximately 10% of legal blindness in the western world. Autoimmune uveitis is driven by activated CD4+ T cells that differentiate into effector T helper cells (Th1, Th2, and Th17) which release proinflammatory cytokines that damage the retina. In this study we investigated the effect of the methionine aminopeptidase 2 (MetAP2) inhibitor, Lodamin, on T cell activation and differentiation. MetAp2 is an enzyme which regulates cellular protein synthesis and is highly expressed in T cells. Lodamin was found to suppress T cell receptor (TCR) mediated T cell proliferation and reduced the production of Th1 and Th17 cells. Further, Lodamin suppressed overall inflammation in the mouse model of experimental autoimmune uveitis (EAU) by a six fold. This effect was attributed in part to a reduction in retinal proinflammatory cytokines, down regulation of MetAP2 expression in purified lymph node CD4+ T cells, and a general normalization of the systemic immune reaction.

Identification of Padi2 as a novel angiogenesis-regulating gene by genome association studies in mice

Recent findings indicate that growth factor-driven angiogenesis is markedly influenced by genetic variation. This variation in angiogenic responsiveness may alter the susceptibility to a number of angiogenesis-dependent diseases. Here, we utilized the genetic diversity available in common inbred mouse strains to identify the loci and candidate genes responsible for differences in angiogenic response. The corneal micropocket neovascularization assay was performed on 42 different inbred mouse strains using basic fibroblast growth factor (bFGF) pellets. We performed a genome-wide association study utilizing efficient mixed-model association (EMMA) mapping using the induced vessel area from all strains. Our analysis yielded five loci with genome-wide significance on chromosomes 4, 8, 11, 15 and 16. We further refined the mapping on chromosome 4 within a haplotype block containing multiple candidate genes. These genes were evaluated by expression analysis in corneas of various inbred strains and in vitro functional assays in human microvascular endothelial cells (HMVECs). Of these, we found the expression of peptidyl arginine deiminase type II (Padi2), known to be involved in metabolic pathways, to have a strong correlation with a haplotype shared by multiple high angiogenic strains. In addition, inhibition of Padi2 demonstrated a dosage-dependent effect in HMVECs. To investigate its role in vivo, we knocked down Padi2 in transgenic kdrl:zsGreen zebrafish embryos using morpholinos. These embryos had disrupted vessel formation compared to control siblings. The impaired vascular pattern was partially rescued by human PADI2 mRNA, providing evidence for the specificity of the morphant phenotype. Taken together, our study is the first to indicate the potential role of Padi2 as an angiogenesis-regulating gene. The characterization of Padi2 and other genes in associated pathways may provide new understanding of angiogenesis regulation and novel targets for diagnosis and treatment of a wide variety of angiogenesis-dependent diseases.

Epsin deficiency promotes lymphangiogenesis through regulation of VEGFR3 degradation in diabetes

Impaired lymphangiogenesis is a complication of chronic complex diseases, including diabetes. VEGF-C/VEGFR3 signaling promotes lymphangiogenesis, but how this pathway is affected in diabetes remains poorly understood. We previously demonstrated that loss of epsins 1 and 2 in lymphatic endothelial cells (LECs) prevented VEGF-C–induced VEGFR3 from endocytosis and degradation. Here, we report that diabetes attenuated VEGF-C–induced lymphangiogenesis in corneal micropocket and Matrigel plug assays in WT mice but not in mice with inducible lymphatic-specific deficiency of epsins 1 and 2 (LEC-iDKO). Consistently, LECs isolated from diabetic LEC-iDKO mice elevated in vitro proliferation, migration, and tube formation in response to VEGF-C over diabetic WT mice. Mechanistically, ROS produced in diabetes induced c-Src–dependent but VEGF-C–independent VEGFR3 phosphorylation, and upregulated epsins through the activation of transcription factor AP-1. Augmented epsins bound to and promoted degradation of newly synthesized VEGFR3 in the Golgi, resulting in reduced availability of VEGFR3 at the cell surface. Preclinically, the loss of lymphatic-specific epsins alleviated insufficient lymphangiogenesis and accelerated the resolution of tail edema in diabetic mice. Collectively, our studies indicate that inhibiting expression of epsins in diabetes protects VEGFR3 against degradation and ameliorates diabetes-triggered inhibition of lymphangiogenesis, thereby providing a novel potential therapeutic strategy to treat diabetic complications.

Abstract 2140: Oral antiangiogenic therapy suppresses xenograft growth and is well tolerated in rats.

Lodamin, the oral formulation of TNP-470, a methionine aminopeptidase 2 (MetAp2) inhibitor, was previously shown to significantly inhibit angiogenesis and suppress the progression of fast growing murine tumors. Lodamins’ unique structure as a polymer micelle composed of poly(ethylene-glycol)-poly(lactic)acid (PEG-PLA) conjugate of TNP-470 is pharmacologically advantageous over the non-conjugated form. It shows better stability, oral availability, solubility in water and importantly, has an improved safety profile (Benny O. Folkman J et al, Nat Biotech, 2008). We now report the successful large scale synthesis of Lodamin, yielding pure and active product composed of mPEG-PLA-TNP-470 conjugate without residual free drug as confirmed by NMR and LC-MS/MS. The safety profile of Lodamin was studied in an acute high dose rat study (4 fold higher than therapeutic dose). Lodamin was administered at 10mg/kg or 60mg/kg via oral gavage while control rats were given vehicle only. After 7 days of daily treatment, no changes in body weight or behavior were observed compared to the control group. On day 8 post treatment, gross pathology of all rats was examined. This data showed Lodamin treatment had no adverse effect on tissue morphology. Lodamin is a potent antiangiogenic drug in numerous in vivo murine models including the corneal micropocket assay, matrigel assay, Delayed-Type Hypersensitivity (DTH) reaction and the choroidal neovascularization (CNV) model (Benny O. Folkman J et al, Nat Biotech, 2008, Benny O. D9Amato R et al, Plos One 2010). We now show that in addition to its dramatic anti-cancer effects on Lewis lung carcinoma and B16F10 melanoma murine tumors, Lodamin suppresses human tumors induced in nude mice. The volume of subcutaneous (s.c) human glioblastoma (U87MG) xenografts were reduced by 70% after 30 days, and 71% after 45 days when administered by gavage or in drinking water, respectively. Human breast tumor (MDA-MB231, s.c) growth was inhibited by 62% after 47 days of administration, and Human hepatocarcinoma (HepG2, s.c) tumor growth was inhibited by 60% after only 12 days. Taken together we have established Lodamin as a potent antiangiogenic drug which retains TNP-470 activity without TNP-470-associated side effects. Lodamin is a safe and efficacious drug in our animal models and hopefully will be reintroduced for the treatment of cancer in patients. Citation Format: Ofra Benny, Lauren Bazinet, Robert D9Amato. Oral antiangiogenic therapy suppresses xenograft growth and is well tolerated in rats. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2140. doi:10.1158/1538-7445.AM2013-2140