Comparative effects of bevacizumab, ranibizumab and pegaptanib at intravitreal dose range on endothelial cells

https://doi.org/10.1016/j.exer.2008.11.011Get rights and content

Abstract

Anti-VEGF therapy proved to be useful against several ocular pathological situations, including choroidal neovascularization and proliferative retinopathies. Ranibizumab (Ran), Pegaptanib (Peg) and Bevacizumab (Bev) are the pharmacological agents more frequently used in clinical practice by intravitreal injection. However, their exact effects on the angiogenic process have not been accurately established in a comparative study. The aim of the present study was to elucidate the precise effects of Ran, Peg and Bev on the multiple steps of the angiogenic process. Human umbilical vein endothelial cells (HUVEC) were incubated with each agent within the clinically established concentration range, or identical amounts of the excipients; cell cytotoxicity, proliferation, apoptosis, migration and vessel assembly were assessed. No cytotoxic effects were found for any of the agents studied at any concentration tested. At the clinical dose, cell proliferation was significantly reduced by Bev and Ran, whereas no difference was observed after Peg treatment. In addition, HUVEC apoptosis was effectively increased by Bev and Ran. Cell migration was reduced after incubation with every agent analyzed, though only reaching statistical significance upon Ran intravitreal dose. At clinical doses, capillary assembly was only affected by Bev. In agreement with these data, the active form of VEGF receptor-2 expression was decreased after incubation with Bev (to 66% of control values), Ran (78%) and Peg (86%) relative to controls. These findings indicate that these three agents display distinct effects on endothelial cells.

Introduction

Angiogenesis is a key aspect in many ocular pathologies that are leading causes of blindness in the world, such as neovascular age-related macular degeneration (AMD), diabetic retinopathy, retinopathy of prematurity (ROP), central retinal vein occlusion and other diseases associated with ischemia and neovascularization (Eichler et al., 2006).

Although angiogenesis is a highly complex and coordinated process requiring multiple receptors and ligands in endothelial cells, vascular endothelial growth factor (VEGF) appears to be a pivotal element required for the process in a variety of normal and pathological circumstances (Ferrara et al., 2004, Ferrara et al., 2006). Since VEGF appears to be the most important activating factor of angiogenesis and vascular permeability, new molecules that bind and inhibit VEGF were developed in recent years.

Pegaptanib sodium (Peg) (Macugen ®, Pfizer), an aptamer that specifically binds extracellular VEGF isoforms that are ≥165 amino acids in size, was the first VEGF-binding drug to be approved for intravitreal treatment of neovascular AMD (Gragoudas et al., 2004, Quiram et al., 2007).

In July 2006, Ranibizumab (Lucentis®, Genentech and Novartis)-, a high-affinity, recombinant, humanized, antigen-binding fragment Fab that neutralizes all active isoforms of VEGF, derived from the same anti-VEGF murine monoclonal antibody that originated Bevacizumab – was also approved by the Food and Drug Administration (FDA) for the treatment of wet AMD. Administered by intravitreal injection, Ranibizumab (Ran) prevents visual loss and improves mean visual acuity in treated patients (Rosenfeld et al., 2006, Brown et al., 2006).

Bevacizumab (Bev) (Avastin ®) was the first humanized anti-VEGF neutralizing antibody approved by the FDA for treatment of metastatic colon cancer (Ferrara et al., 2004). Additionally, Bev combined with chemotherapy has been used in clinical trials for several types of cancer (Shojaei and Ferrara, 2007). More recently, following the first intravitreal application of Avastin® in 2005 (Rosenfeld et al., 2005), the off-label administration of intravitreal Bev in the treatment of exsudative AMD is now widely used (Avery et al., 2006, Spaide et al., 2006), with positive visual results, at a lower price. There are no head to head studies that compare the efficacy of the three different molecules in the treatment of ocular neovascular pathologies (Steinbrook, 2006). On the other hand, the efficacy of the three drugs in other pathologies such as diabetic macular edema (DME), proliferative diabetic retinopathy, central retinal vein occlusion and ROP is being investigated. The FDA has not approved the use of any of the three drugs for the treatment of other angiogenic ocular diseases apart from wet AMD. However, their off-label use has started a new era in the treatment of neovascular blinding ophthalmologic diseases.

Although largely used in clinical practice, there are no studies comparing the three agents in order to evaluate their relative safety and efficacy in vivo and only few reports compare the molecules in vitro.

In the present work, we evaluate the precise effects of Ran, Peg and Bev in several steps of angiogenesis. Human umbilical vein endothelial cells (HUVEC) were incubated with the three agents at concentrations within the clinically established range, or identical amounts of the excipients; cell cytotoxicity, proliferation, apoptosis, migration and vessel assembly were assessed. Additionally, the expression of the phosphorylated (active) form of VEGFR2 in HUVEC cultures previously treated with Bev, Ran or Peg was assessed by Western blotting assays.

Section snippets

Cell culture experiments

Human umbilical vein endothelial cells (HUVECs) were obtained from ScienceCell Research Labs (San Diego, USA). Cells were used between passages 3 and 8. HUVECs were cultured in M199 medium (Sigma–Aldrich, Portugal) supplemented with 20% FBS (Invitrogen Life Technologies, Scotland, UK), 1% penicillin/streptomycin (Invitrogen Life Technologies, Scotland, UK), 0.1% heparin (Sigma–Aldrich, Portugal) and 0.1% endothelial cell growth supplement (ECGS) (Sigma–Aldrich, Portugal), and maintained at 37 °C

Effect of anti-VEGF agents on cell viability

We first attempted to evaluate whether the three anti-VEGF agents developed cytotoxic effects. HUVEC were cultured until 90% confluency and cytotoxicity was assessed by MTT assay. As observed in Fig. 1, cell viability was not affected by Bev, Ran or Peg at any concentration tested, as compared to excipient-treated cell cultures (controls). MTT assays were performed in highly confluent cell cultures, thus presenting an already slow replication rate. This ensures that the possible toxic effect is

Discussion

Although widely used as intravitreal therapies for many vasoproliferative ocular diseases worldwide, there are only few and limited comparative studies in the literature to evaluate the relative safety and efficacy of Peg, Ran and Bev in vitro. In the current study, we aimed at an overview of the effects observed after treating HUVEC with these three anti-VEGF agents on the multiple steps that comprise angiogenesis. We first evaluated whether these agents induced cytotoxicity in confluent HUVEC

Acknowledgements

The authors would like to thank Professor Isabel Azevedo for the helpful discussions. This study was partially funded by FCT (POCI/BM/55556/04, Feder and Programa Comunitário de Apoio), by University of Porto, undergraduation project (PG/07) and by “Sociedade Portuguesa de Oftalmologia”.

References (27)

  • J. Lowe et al.

    Ranibizumab inhibits multiple forms of biologically active vascular endothelial growth factor in vitro and in vivo

    Exp. Eye Res.

    (2007)
  • F. Shojaei et al.

    Antiangiogenesis to treat cancer and intraocular neovascular disorders

    Lab. Invest.

    (2007)
  • R.S. Apte

    Pegaptanib sodium for the treatment of age-related macular degeneration

    Expert Opin. Pharmacother.

    (2008)
  • R.L. Avery et al.

    Intravitreal bevacizumab (Avastin) for neovascular age related macular degeneration

    Ophthalmology

    (2006)
  • D. Brown et al.

    Ranibizumab versus verteporfin for neovascular age-related macular degeneration

    N. Engl. J. Med.

    (2006)
  • W. Eichler et al.

    Antineovascular agents in the treatment of eye diseases

    Curr. Pharm. Des.

    (2006)
  • EMEA, 2005....
  • N. Ferrara et al.

    Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer

    Nat. Rev. Drug Discov.

    (2004)
  • N. Ferrara et al.

    Development of ranibizumab, an anti-vascular endothelial growth factor antigen binding fragment, as therapy for neovascular age-related macular degeneration

    Retina

    (2006)
  • J. Gaudreault et al.

    Preclinical pharmacokinetics of ranibizumab (rhuFabV2) after a single intravitreal administration

    Invest. Ophthalmol. Vis. Sci.

    (2005)
  • E. Gragoudas et al.

    Pegaptanib for neovascular age-related macular degeneration

    N. Engl. J. Med.

    (2004)
  • A.K. Klettner et al.

    Comparison of bevacizumab, ranibizumab and pegaptanib in vitro: efficiency and possible additional pathways

    Invest Ophthalmol. Vis. Sci.

    (2008 Oct)
  • S. Michels et al.

    Promising new treatments for neovascular age-related macular degeneration

    Expert Opin. Investig. Drugs

    (2006)
  • Cited by (0)

    View full text