horizon the on agents perspectives: • 53Futurechapter

MAMMALIAN TARGET OF

RAPAMYCIN INHIBITOR

Rapamycin (sirolimus) is a macrolide antibiotic that is produced naturally by Streptomyces hygroscopicus. Rapamycin binds to FKBP12. The resulting complex binds to the mammalian target of rapamycin (mTOR), inhibiting the function of mTOR in the mTORC1 complex.22 The mammalian target of rapamycin is a key component in a cell’s response to insulin, insulin-like growth factors, nutrient levels, hypoxia, and redox status. mTOR forms the complexes mTORC1 and mTORC2, where only mTORC1 is susceptible to regulation by rapamycin. mTORC1 promotes mRNA translation by phosphorylating and activating the p70-S6 kinase 1 (S6K1) and phosphorylating and inactivating eukaryotic initiation factor 4E (eIF4E) binding protein 1(4E-BP1), which is a repressor of mRNA translation.23 By a separate mechanism, in the presence of activated mTOR, the regulatory-associated protein of mTOR (Raptor), which is part of the mTORC1 complex, interacts with HIF-1α to activate it.24 Thus sirolimus has an inhibitory effect on HIF-1α. Rapamycin inhibits mTORC1 activity and has antiproliferative, antiangiogenic, and immunosuppressive functions.25 Rapamycin (Macusight) underwent phase I testing in neovascular AMD as a sustained-release intravitreal or subconjunctival injection in a dose escalation study. Currently, Macusight is sponsoring a phase II trial that is randomizing 120 patients between three different doses of subconjunctival sirolimus and placebo. The main outcomes will be visual acuity and central macular thickness. The National Eye Institute is also conducting a study assessing the role of immunomodulatory agents in combination with routine care using antiangiogenic therapies for the treatment of sub­ foveal CNV secondary to AMD. Twenty patients will be randomly assigned to one of four study arms. Patients will receive intravenous daclizumab, intravenous infliximab, oral sirolimus, or no study medication. The study will assess recurrence of neovascularization, as well as changes in visual acuity and retinal thickening as measured by OCT over a 24-week interval.

A phase I/II study of ocular sirolimus is being conducted. Patients will be randomized between three subconjunctival injections of 1320 µg or three intravitreal injections of 352 µg of sirolimus. Visual acuity will be assessed over 180 days, and safety endpoints over 1 year.26

The role of sirolimus in the treatment of DME is also being investigated in two trials. Sirolimus is an excellent candidate for treating DME due to its anti-inflammatory action, effect on HIF-1α levels, and reduction of vascular permeability. The National Eye Institute is evaluating the efficacy of subconjunctival injections of 440 µg of sirolimus at baseline and at 2 months on visual acuity and macular edema, as measured by OCT in 5 patients.27

SMALL INTERFERING RNA

PF-04523655 (Quark/Pfizer) is a small interfering RNA inhibitor of RTP801. RTP801 is a hypoxia-inducible gene which acts via the TSC1/ TSC2 tumor suppressor complex to inhibit mTOR.28,29 PF-04523655 has been demonstrated to be efficacious in inhibiting the development of CNV in a murine laser-induced CNV model. Moreover, the drug appeared to have an additive effect when used in combination with pegaptanib or an antibody against mouse VEGF. The drug has been studied in mouse, rat, rabbit, and monkey systems, and a human phase I study has been performed. A phase II prospective, randomized, doseranging study of PF-04523655 in comparison with ranibizumab for the treatment of subfoveal CNV secondary to AMD is currently recruiting patients. The study has two stages, where the first will determine whether an initial injection of ranibizumab is required before starting PF-04523655. The structure of the second stage will depend on the results of the first stage. If a “first-line” design is used, 160 patients will be randomized 1 : 1 : 1 : 1 between three different doses of PF-04523655 or ranibizumab, and will receive the study drug on a monthly basis until week 44 of the trial. If an “induction” design is implemented, all patients will receive an initial treatment with ranibizumab, and will then receive monthly injections of the study drug to which they have

been randomized. The primary endpoint of the study will be best corrected visual acuity at 48 weeks. Secondary endpoints will be changes in fluorescein angiography and OCT parameters, and requirement for rescue therapies. Other pharmacologic agents that employ small interfering technology, such as siRNA-027 and bevasiranib, have been described elsewhere in this book.

BIOACTIVE LIPIDS

There is increasing recognition that many classes of lipids, especially metabolites of cell membrane components, have a role in signal transduction and in the regulation of multiple cellular processes. Lipids with these roles are known as “bioactive lipids.” The sphingolipids, formed from the condensation of serine and palmitate, are of particular interest in the regulation of the vascular endothelium. The sphingolipid sphingomyelin is an important component of cellular membranes. It is synthesized from and broken down to ceramide, which in turn is broken down by ceramidase to yield sphingosine, and then phosphorylated by the sphingosine kinases (SK1 and SK2) to yield sphingosine 1- phosphate (S1P). S1P is the ligand for five different cell surface S1P receptors. S1P, S1P receptors, and sphingosine kinases have all been demonstrated to be present in the eye.30

S1P is intimately involved in regulation of endothelial function. S1P1 receptors are required for recruitment of vascular smooth-muscle cells and pericytes during mouse embryonic vessel formation.31 S1P induces proliferation and migration of human endothelial cells,32 and regulates basal and angiopoietin-1-mediated endothelial permeability.33 There is extensive cross-talk between S1P and VEGF receptors: S1P can activate VEGFR-2 in the absence of VEGF; VEGF sensitizes endothelium to the action of S1P by up-regulating its receptors; activated PDGF receptor activates sphingosine kinase 1 to the cell membrane, activating S1PR1.

S1P has a role in animal models of retinal disease. In mice, an anti-S1P monoclonal antibody inhibits the development of laser-induced CNV.30 S1P2 receptor knockout mice demonstrate reduced angiogenesis in the oxygen-induced retinopathy model.34 Systemic treatment with a smallmolecule inhibitor of sphingosine kinase significantly reduced retinal fluorescein leakage in diabetic rats.35 A monoclonal antibody against S1P has been humanized and is in phase I trials as an antiangiogenic agent in cancer treatment. A phase I trial for neovascular AMD is planned. If the antibody is well tolerated, its function in human diabetic retinopathy will also be of interest.

NONNEOVASCULAR AGE-RELATED MACULAR DEGENERATION

COMPLEMENT INHIBITORS

The complement cascade is part of the innate immune system.36 The complement system consists of a cascade of cleavage enzymes, regulatory molecules, and the components of the membrane attack complex. The membrane attack complex forms a pore through the cellular surface membranes, and if sufficient numbers of pores are present, cell death will ensue. Many of the cleavage products of the complement cascade have biological functions in addition to their enzymatic role in the cascade. Complement component C3b (where the complement components are designated by “C” followed by a number and then, where appropriate, “a” or “b” to indicate the cleavage products) enhances opsonization. C5a is chemotactic for inflammatory cells, and activates macrophages. C3a and C5a trigger mast cell degranulation. C5b initiates the formation of the membrane attack complex.

Activation of the complement cascade may occur via one of three pathways. The classical pathway is initiated when complement component C1q binds to IgM– or IgG–antigen complexes, or when it binds directly to the surface of a pathogen. The lectin pathway is similar: it is activated by the binding of either ficolins or mannose-binding lectin to the surface of a pathogen. The alternative pathway is triggered by the