Figure 16.1 Pegaptanib structure and target binding. A. Sequence and predicted secondary structure of pegaptanib. 2′-O-methylated purines are shown in red, 2′-fluorine-modified pyrimidines are shown in blue, and unmodified ribonucleotides are shown in black. The site of attachment of a 40-kDa PEG moiety is shown. B. Interaction between the 55-amino-acid heparin-binding domain of VEGF₁₆₅ and pegaptanib. (From Ng EW, Shima DT, Calias P, et al. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat Rev Drug Discov. 2006;5(2):123–132.)

Aptamers are created using a process known as the systematic evolution of ligands of exponential (SELEX) enrichment process (9). Through this process, 10¹⁴ aptamers are produced in a test tube and then screened for their ability to bind or interfere with various target molecules (9). Using VEGF₁₆₅ as the target, SELEX methodology was performed in three separate approaches at NeXstar Pharmaceuticals, ultimately producing the aptamer that became pegaptanib. Aptamers that blocked the actions of VEGF in vitro were first described in 1994 (10) followed by the use of amino-substituted nucleotides to improve the resistance of anti-VEGF aptamers to nuclease attack in 1995 (11) and, subsequently, the use of additional substitutions to further improve stability and affinity in 1998 (12). In the latter work, three stable, high-affinity candidate anti-VEGF aptamers were characterized, one of which was selected for development as pegaptanib. In a process called pegylation, the addition of a 40-kDa polyethylene glycol (PEG) moiety to the 5′ end of the aptamer improved bioavailability (12). Pegylation is the process of covalent attachment of PEG polymer chains to another molecule, normally a drug or therapeutic protein (13). This covalent attachment can “mask” the agent from the host’s immune system (reducing immunogenicity and antigenicity) and increase the hydrodynamic size (size in solution) of the agent, which prolongs its terminal half-life by reducing clearance from the eye (14).

The selectivity of pegaptanib derives from its interaction with cysteine-137, an amino acid that is contained within the 55 amino acid heparin-binding domain of VEGF, which is not present in VEGF₁₂₁ (15); the interaction between pegaptanib and this domain is shown in Figure 16.1B (16).

CHEMICAL STRUCTURE

The molecular formula for pegaptanib sodium is C₂₉₄H₃₄₂ F₁₃N₁₀₇Na₂₈O₁₈₈P₂₈[C₂H₄O]n (where n is approximately 900), and the molecular weight is 49 kDa (17). The chemical structure of pegaptanib sodium is depicted in Figure 16.2.

RATIONALE FOR CLINICAL USE

The VEGF family is a group of secreted proteins that include VEGF-A, -B, -C, -D, and placental growth factor (PlGF). These factors selectively bind and activate receptors located primarily on the surface of vascular endothelial cells. Among these, it is well known that VEGF-A acts as a key regulator of pathologic vessel growth and vascular permeability in ocular neovascular disease such as AMD (18,19). VEGF is produced by many cells types in the retina (20,21), but the underlying causes for its elevation during ocular neovascularization are not known. It has been suggested that VEGF can be up-regulated by plasminogen activator (22), endothelial nitric oxide synthase (23), and, most of all, hypoxia (24), which further contributes to the pathogenesis of ocular vascular disease. Several biologically active isoforms have been identified depending on the site of splicing of the VEGF gene: VEGF_{121−165−189}, and ₋₂₀₆. The isoform VEGF₁₆₅ is thought to be the most associated with pathologic ocular neovascularisation (25). Of the other isoforms, VEGF₁₈₉ and VEGF₂₀₆ are highly basic, are heparin binding, and exist primarily as matrix-bound forms. VEGF₁₂₁, lacking the heparin-binding domain, is freely secreted (22). Pegaptanib sodium was developed to selectively inhibit the isoform VEGF₁₆₅. In this way, the choice of aptamer was based on the characteristic of having high affinity and specificity for VEGF₁₆₅. In fact, preclinical studies in mouse models of pathologic ocular neovascularisation demonstrate the effectiveness of pegaptanib sodium at inhibiting the activity of VEGF₁₆₄ (the murine homologue to the human isoform VEGF₁₆₅) (26,27). However, one of the main concerns during drug development was whether or not the inhibition of VEGF₁₆₅ could inhibit not only pathologic neovascularisation but physiologic vasculogenesis and vascular homeostasis. The studies mentioned above showed that VEGF₁₆₄ was selectively increased in pathologic neovascularisation, and blocking it results in inhibition of pathologic angiogenesis and vascular permeability without damage to normal vasculature (26,27).

The rationale behind this approach is that eliminating the stimulus for the formation of abnormal blood vessels, one can eliminate the unwanted effects of the abnormal blood vessels (i.e., intraretinal, subretinal, and sub–retinal pigment epithelium [RPE] fluid accumulation and hemorrhage) without the “collateral” damage induced by thermal laser photocoagulation or PDT. One of the main advantages of anti-VEGF treatment over thermal laser or PDT is that normal retina, RPE, and choroid are spared from damage (28).

CLINICAL PHARMACOLOGY

Pharmacokinetics

Absorption

In animals, pegaptanib is slowly absorbed into the systemic circulation from the eye after intravitreous administration. The rate of absorption from the eye is the rate-limiting step in the disposition of pegaptanib in animals and is likely to be the rate-limiting step in humans, too (17). Vitreous levels appear to follow an apparent first-order elimination process. Preclinical studies determined the terminal half-life in the vitreous to be 83 hours in a rabbit model (6) and 94 hours (29) in a rhesus monkey model. The estimated plasma terminal half-life was 84 hours, and plasma concentration of the drug diminished via an apparent first-order elimination process as well. In humans, a mean maximum plasma concentration of about 80 ng/mL occurs within 1 to 4 days after a 3-mg monocular dose (10 times the recommended dose) (17). At doses less than 0.3 mg per eye, pegaptanib sodium plasma concentrations are not likely to exceed 10 ng/mL, which is more than 100 times less than the concentration observed in the nonclinical toxicology studies at the “no observed adverse effect level” doses (17). Circulating levels of pegaptanib sodium seen 4 to 6 weeks after an intravitreous 0.3-mg dose were below the lower limits of quantification (8 ng/mL) of the assay (17).

Metabolism

Based on preclinical data, pegaptanib is metabolized by endo- and exonucleases (17).

Excretion

After intravitreous and intravenous administration of radiolabeled pegaptanib to rabbits, the highest concentrations of radioactivity were obtained in the kidney. In rabbits, the component nucleotide 2′-fluorouridine is found in plasma and urine after single radiolabeled pegaptanib intravenous and intravitreous doses (17). However, dose adjustment for patients with renal impairment is not needed when administering the 0.3-mg dose (17).

Pharmacodynamics

Preclinical studies established that pegaptanib inhibited VEGF binding and VEGF-mediated cell signaling in cultured endothelial cells and that pretreatment of cells with pegaptanib inhibited proliferative responses only to VEGF₁₆₅, but not to VEGF₁₂₁ (30). In addition, pegaptanib dramatically inhibited VEGF₁₆₅-induced vascular permeability (6,12).

DRUG DELIVERY AND DOSING

Pegaptanib sodium is available in a single-use, pre-filled 1-mL glass syringe. Pegaptanib sodium (3.47 mg/mL solution) in a volume of 90 μL is delivered intravitreally via the pars plana for a total intraocular dose of 0.3 mg (17,31,32). The drug is packaged free of preservative, although pH buffers are a component of the clear, colorless solution (17).

PIVOTAL TRIALS OF CLINICAL EFFICACY

After phase I and phase II clinical trials demonstrated that pegaptanib was a safe drug (6,7), the hypothesis that pegaptanib would be effective in all types of CNV secondary to AMD was evaluated in two (EOP1003 and EOP1004) identically designed, concurrent, prospective, multicenter, randomized, double-masked, sham-controlled, dose-ranging pivotal trials: the VEGF Inhibition Study in Ocular Neovascularization (VISION) trials. The results were published by the VISION Clinical Trial Group in 2004 (31).

Study Design

Patients with subfoveal CNV due to neovascular AMD were treated every 6 weeks for 48 weeks with either a sham intravitreal injection or intravitreal pegaptanib sodium injection at doses of 0.3, 1, or 3 mg. Pharmacokinetic studies indicated that in order to maintain the intravitreal angiogenesis inhibitory concentration at therapeutic levels, pegaptanib doses ≥0.3 mg would need to be administered at least every 6 weeks (Fig. 16.3). The prespecified study end point was 54 weeks (6 weeks following the last study injection). The use of verteporfin PDT was allowed, in either sham injection groups or pegaptanib injection groups, at the treating physician’s discretion if the FDA labeling indications for verteporfin treatment were met throughout the study.

At the end of 54 weeks, patients originally randomized to receive pegaptanib were rerandomized (1:1) to continue pegaptanib for an additional 48 weeks or to discontinue treatment; patients originally randomized to the sham group were rerandomized (1:1:1:1:1) to either continue in the sham group, to discontinue sham, or to be treated with one of the three pegaptanib doses. The purpose of rerandomization during the 2nd year of the study was to determine whether chronic treatment beyond 1 year was necessary and to identify any additional safety concerns of chronic treatment. The inclusion and exclusion criteria of the study are summarized in Table 16.1.

Outcome Measures

The prespecified efficacy end point was the proportion of patients who lost less than 15 Early Treatment Diabetic Retinopathy Study (ETDRS) letters from baseline to 54 weeks. This represents a doubling of the visual angle, which most ophthalmologists and patients consider as a meaningful change in vision. Secondary outcomes were the proportion of patients gaining ≥15 ETDRS letters from baseline to 54 weeks, the proportion of patients who experienced maintenance or gain of ≥0 letters from baseline to 54 weeks, and the change in mean visual acuity at 6, 12, and 54 weeks.

Clinical Efficacy Data

A total of 1,208 patients were randomly assigned in the two trials, of which 1,186 patients received at least one treatment and were included in 1-year efficacy analyses (622 patients included in EOP1003 and 586 patients included in EOP1004). During year one, 7,545 intravitreal injections of pegaptanib and 2,557 sham injections were administered. An average of 8.5 injections per patient was administered, and approximately 90% of patients in each group completed year 1 of the study.

The individual and combined data from the two clinical trials demonstrated that pegaptanib sodium provided a clear treatment benefit for doses of 0.3 or 1 mg when given intravitreally every 6 weeks for 54 weeks (data presented are from the combined analysis of the individual EOP1003 and EOP1004 trials). The greatest treatment benefit was seen with the lowest studied dose, 0.3 mg (17,31,32). The 0.3-mg dose demonstrated a significantly greater proportion (70%, combined analysis, P < 0.0001) of patients achieving the primary efficacy end point of a loss of less than 15 ETDRS letters of visual acuity at the end of 54 weeks compared with the sham injection group (55%). So far, there is no explanation for this phenomenon that the lowest dose was the most efficient. Figure 16.4 summarizes the results regarding the primary end point.

With regard to mean visual acuity loss, pegaptanib was significantly of benefit compared with sham from as early as the first study visit at 6 weeks. This early benefit was sustained at every visit through the 54-week follow-up (Fig. 16.5) and continued during the 2nd year of the trial (33). Moreover, pegaptanib has shown efficacy as maintenance therapy after any other treatments for neovascular AMD (34).

Maintenance or gain of visual acuity was seen in 33% of the 0.3-mg pegaptanib group compared with 23% in the sham group at the study end point (P = 0.0032). Six percent of patients in the 0.3-mg pegaptanib group gained three lines of vision or more compared to 2% in the sham group (P = 0.0401). On the other hand, 22% of patients in the sham group had a severe vision loss (≥30 ETDRS letters) compared to 10% of patients in the 0.3-mg pegaptanib group (P < 0.0001) (Fig. 16.6).

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