KEY FEATURES

Aptamers are chemically synthesized nucleic acid or peptide oligomers that bind a specific target molecule, thus acting like “chemical antibodies.” The first and only aptamer therapeutic to be approved by the Food and Drug Administration (FDA) was pegaptanib sodium (Macugen, EyetechInc.,PalmBeachGardens,FL),whichwasapprovedinDecember, 2004, and is indicated for the treatment of all angiographic subtypes of neovascular age-related macular degeneration (AMD). Pegaptanib’s pharmacologic characteristics as well as its development for the treatment of vascular diseases of the eye are described in this chapter.

INTRODUCTION AND HISTORY

Pegaptanib is a selective anti-vascular endothelial growth factor (VEGF) aptamer that acts in the extracellular space to inhibit the 165 isoform (VEGF165), while sparing VEGF121.1 Aptamers (from the Latin aptus, meaning “to fit”) are oligonucleic acid or peptide molecules that bind a specific target molecule.2 Intramers are aptamers that act on intracellular targets.3 Aptamers with a high specificity for virtually any protein target can be selected using the in vitro selection process known as systematic evolution of ligands by exponential enrichment (SELEX), in which a population of randomized RNA or DNA oligonucleotides (typically 20–40 nucleotides long) is incubated with the protein target of interest.4 Aptamers that bind to the target are selected, amplified by reverse transcriptase-polymerase chain reaction, and then reselected in an iterative process repeated as needed to achieve desired affinity and specificity. Selected aptamers can then be sequenced and chemically synthesized.

Using the SELEX process, aptamers blocking VEGF in vitro were first described in 1994 by scientists at NeXstar Pharmaceuticals, who were the first to demonstrate the feasibility of an aptamer-based approach to inhibiting neovascularization.5 These experiments were followed in 1995 by the use of NH2-substituted nucleotides to improve the resistance of anti-VEGF aptamers to nuclease attack6 and, subsequently, the use of 2′-F-substituted pyrimidines to improve affinity further in 1998.7 The addition of a 40-kDa polyethylene glycol (PEG) moiety to the 5’ end of this aptamer improved its ability to block VEGF-mediated vascular permeability in a guinea pig assay, probably due to slowed tissue diffusion.7 Further experiments demonstrated that an optimized antiVEGF aptamer, subsequently named pegaptanib sodium, inhibited VEGF binding and VEGF-mediated cell signaling in cultured endothelial cells, and that pretreatment of cells with pegaptanib inhibited proliferative responses to VEGF165, but not VEGF121.8

In follow-up work, pegaptanib was found to be stable in human plasma,9 paving the way for its evaluation in animal models and in clinical studies. Following intravitreous injection, biologically active pegaptanib could be detected in the vitreous humor of monkey eyes for at least 28 days following a single 0.5-mg dose and was determined to be eliminated from the eye through plasma clearance.10 No toxicities related to pegaptanib were observed following systemic or ocular administration in monkeys.10 These preclinical studies established the

biological and pharmacokinetic properties of pegaptanib, thus providing the groundwork for clinical trials of pegaptanib in patients with neovascular AMD, diabetic retinopathy, and retinal vascular occlusive disease. Pegaptanib sodium (Macugen) was approved by the FDA in December of 2004, and was both the first approved aptamer therapeutic and the first approved pharmacotherapy for neovascular AMD.

PHARMACOLOGY

Pegaptanib sodium injection is a sterile, clear, preservative-free aqueous solution supplied in a single-dose, prefilled syringe and is formulated as a 3.47 mg/ml solution, measured as the free acid form of the oligonucleotide.11 The active ingredient is 0.3 mg of the free acid form of the oligonucleotide without PEG, in a nominal volume of 90 µl. Pegaptanib is formulated to have an osmolality of 280–360 mOsmol/kg, and a pH of 6–7.

Pegaptanib sodium is a covalent conjugate of an oligonucleotide of 28 nucleotides in length that terminates in a pentylamino linker, to which two 20-kDa monomethoxy PEG units are covalently attached via the two amino groups on a lysine residue. The molecular formula for

pegaptanib sodium is C294H342F13N107Na28O188P28[C2H4O]n (where n is approximately 900) and the molecular weight is 49 kDa.11

PHARMACOKINETICS

Pegaptanib exhibits “flip-flop” kinetics, a phenomenon wherein the drug remains in the eye for a significant amount of time, is slowly cleared into the systemic circulation, and then is relatively rapidly cleared from the circulation.12 The pharmacokinetics of pegaptanib following intravitreous injection were profiled in a study of 147 subjects with neovascular AMD.13 Either 1 or 3 mg of pegaptanib sodium per study eye was administered every 6 weeks for 54 weeks. For the 1-mg dose, mean maximal plasma concentrations ranged from 20 to 24 ng/ ml, and pegaptanib was measurable (>8 ng/ml) in the plasma for up to 1 week after injection. The mean apparent terminal plasma half-life, determined from the 3-mg group, was 10 (±4) days. There was no plasma accumulation with administration of repeated doses. Additionally, no serum antibodies against pegaptanib were detected.13

Regarding ocular distribution, pegaptanib penetrates all retinal layers.14 Twenty-four hours following intravitreal injection, labeled pegaptanib can be identified in both the outer nuclear layer and photoreceptor layers, with very little staining within the nuclear layers of the retina. Regarding metabolism and excretion, based on preclinical data, pegaptanib is metabolized by endoand exonucleases and is eliminated as parent drug and metabolites primarily in the urine.11

Plasma concentrations do not appear to be affected by age or gender, but have not been studied in patients under the age of 50. Dose adjustment for patients with renal impairment or weighing greater than 39 kg is not needed when administering the 0.3-mg dose. Pegaptanib has not been studied in patients with hepatic impairment or requiring hemodialysis.11