alternative approach to obtain sustained release of macromolecules such as proteins. The cell reservoir within an implant may be an efficient mechanism to slowly release the drug over prolonged periods. However, the long-term safety of any other factors released by these implants has yet to be ascertained.

17.5  Case Studies

Vascular endothelial growth factor (VEGF) is a vasoactive cytokine capable of increasing blood vessel permeability and proliferation. The VEGF family consists of peptides VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and placental­ growth factor (PIGF); all have similar structural domains, but each have different biological and physical properties (Ferrara 2004). Alternative exon splicing of the VEGF-A gene generates eight isoforms and the predominant isoforms are VEGF121, VEGF165 (most predominant isoform), VEGF189, and VEGF206. VEGF plays a key role in angiogenesis and vasculogenesis (Ferrara and Gerber 2001; Takahashi and Shibuya 2005) during embryonic and early postnatal stage. It acts as a survival factor, vasodilator (Ferrara and Gerber 2001) and has a role in glomerulogenesis, renal glomerular capillary function (Eremina et al. 2003), and the female reproductive cycle (Ferrara and Gerber 2001; Takahashi and Shibuya 2005). In pathological conditions, it plays an important role in wound healing (Nissen et al. 1998) including PDR and DME.

Pegaptanib:  Pegaptanib (Macugen®; Eyetech Pharmaceuticals, New York, USA) is a PEGylated ribonucleic acid (RNA) aptamer that binds and neutralizes human VEGF165 (Gragoudas et al. 2004). It is made up of single-stranded nucleic acid that is synthesized chemically and PEGylated with two 20 kDa PEG molecules attached at each end. Its molecular weight is approximately 50 kDa. It has a unique threedimensional structure that allows it to selectively inhibit VEGF165, which has role in pathological conditions such as wet AMD. In 2004, pegaptanib was approved by the US FDA for the treatment of all forms of neovascular AMD. It is supplied in a single dose prefilled syringe containing 0.3 mg of the drug in 90 mL of injectable volume. Intravitreal injection of pegaptanib in human patients of 0.3 mg every 6 weeks for 2 years showed delayed neovascular AMD progression in comparison to controlled patients (Gragoudas et al. 2004; Donati 2007). Its vitreous half-life is approximately 4 days and the therapeutic level is maintained for 6 weeks after single injection. Pegaptanib injection, every 6 weeks, significantly reduced mean visual acuity loss by approximately 50% in patients having subfoveal CNV as confirmed by the VEGF inhibition study in ocular neovascularization (VISION). Patients with AMD were reported to gain visual benefits when 0.3 mg of pegaptanib was administered intravitreally every 6 weeks for 2 years; that is, visual acuity was maintained compared to those patient who received standard of care or were discontinued from therapy (Chakravarthy et al. 2006). A clinical study confirmed that pegaptanib is systemically and ocularly safe (Cunningham et al. 2005). However, adverse effects associated with pegaptanib included anterior chamber inflammation,

conjunctival hemorrhage, and ocular discomfort, but these adverse effects were mild to moderate and were often due to injection procedure and not due to the drug itself. Further, a rise in intraocular pressure (IOP) was reported due to the intravitreal injection, but the IOP diminished within an hour of injection. Other adverse effects included about 1% incidence of endophthalmitis, retinal detachment, and iatrogenic traumatic cataract, and about 15% incidence of retinal arterial and venous thrombosis. Despite these side effects, pegaptanib is clinically safe up to 0.3 mg dose (Apte et al. 2007).

Bevacizumab:  Bevacizumab (Avastin®; Genentech, California, USA) is a full length humanized antibody (148 kDa) from mouse monoclonal antibody expressed in Chinese hamster ovary cells. It can inhibit all forms of VEGF (Ferrara et al. 2004). Its vitreal half-life is 5.6 days (Bakri et al. 2007b). It has been approved for systemic delivery of colorectal cancer in 2004, but it is also used off-label for the treatment of AMD-related CNV (Hurwitz et al. 2004). Typically it is administered as intravenous infusion with a dose ranging from 5 to 10 mg/kg body weight (infusion time: 1 h) every 2 weeks in combination with other drugs such as 5-flurouracil for treating metastatic colorectal cancer. It blocks all forms of VEGF and hence it might impair both physiological and pathological neovascularization, especially after systemic administration. Intravitreal injections of bevacizumab (1.25 mg in 0.05 mL every month) are capable of preventing ocular angiogenesis (Rosenfeld et al. 2005; Avery et al. 2006; Iliev et al. 2006; Spaide et al. 2006). Patients with neovascular AMD reported a gain in visual acuity by 2.2 lines in 6 months when treated with three intravitreal injections of 1.0 mg of bevacizumab every 4 weeks (Weigert et al. 2008). Similar to other anti-VEGF therapies, bevacizumab is also associated with ocular complications such as uveitis (0.09%), endophthalmitis (0.16%), and retinal detachments (0.16%). Further it can result in acute systemic blood pressure elevation (0.59%) and even death (0.4%) (Arevalo et al. 2007). Triple therapy with verteporfin PDT, bevacizumab, and dexamethasone also showed a significant improvement in the visual acuity after a single cycle of treatment in patients with CNV (Augustin et al. 2007).

Ranibizumab:  Ranibizumab (Lucentis®; Genentech, California, USA), a recombinant humanized antigen Fab fragment (48 kDa) of mouse monoclonal antibody, has one binding site for VEGF (Ferrara et al. 2006). It is a globulin G1K isotype monoclonal antibody fragment. It is expressed in the Escherichia coli expression system. It blocks all forms of VEGF and hence it might impair both physiological and pathological neovascularizations, resulting in increased risk for systemic side effects. In 2006 it was approved for treatment of wet AMD by the US FDA. Its vitreal half-life is 2.88 days in rabbit eye (Bakri et al. 2007a). It is three to sixfold more potent at inhibiting VEGF than bevacizumab (Chen et al. 1999; Heier et al. 2006). A phase III clinical trial was done to evaluate the safety and efficacy of intravitreal injections of ranibizumab (Rosenfeld et al. 2006). A dose of 0.5 mg in 0.05 mL was given monthly to patients with classic CNV secondary to AMD. Ranibizumab effectively increased the visual baseline. Another clinical trial ANCHOR (the anti­ -VEGF antibody for the treatment of predominantly classic CNV in AMD) was conducted in 2006 and it confirmed that ranibizumab was

moderately able to prevent­ the vision loss in neovascular AMD (Rosenfeld et al. 2006). Repeated intravitreal injections of ranibizumab were determined to be well tolerated by three clinical trials: MARINA (Minimally Classic/Occult Trial of the Anti­ -VEGF Antibody Ranibizumab in the Treatment of Neovascular Age-Related Macular Degeneration), PIER (Phase I AMD, Multicenter, Randomized, DoubleMasked, Sham Injection-Controlled Study of the Efficacy and Safety Ranibizumab), and ANCHOR. Transient subconjunctival hemorrhage, minor intraocular inflammation, and transient elevated IOP are few of the minor side effects that have been reported (Heier et al. 2006; Rosenfeld et al. 2006).

VEGF Trap-Eye:  VEGF Trap-Eye (Aflibercept®, Regeneron Pharmaceuticals, New York, USA) is a 110-kDa fusion protein composed of an extracellular VEGF receptor sequence (VEGF1 and VEGF2) fused with a fragment crystallizable (Fc) region of human IgG1 (Saishin et al. 2003). It binds to all VEGF isoforms more tightly than any other anti-VEGF agent (about 140 times higher than ranibizumab). It also has a longer intravitreal half-life compared to ranibizumab due to its larger size. It is predicted to exhibit a longer duration of activity than ranibizumab at similar doses (Stewart and Rosenfeld 2008). It is predicted that VEGF Trap-Eye requires less frequent administration and hence, there will be multiple advantages including lower medicinal cost, less frequent injections, improved patient compliance, and fewer physician appointments (Stewart and Rosenfeld 2008). Intravenous infusion of VEGF Trap-Eye seems to decrease the retinal thickness in a dose-dependent manner (Nguyen et al. 2006). A single intravitreal infusion of VEGF Trap-Eye (1.0 mg/kg body weight, infusion time – 1 h) improved the visual acuity or at least stabilized the visual acuity in 95% of patients after 6 weeks (Nguyen et al. 2006). In addition, a significant reduction in central retinal thickness was seen after 8 weeks of injection. Currently, phase III trials are being conducted.

Ciliary neurotrophic factor:  CNTF is a 64-kDa protein hormone, nerve growth factor, and is also a survival factor for neurons. It has recently been shown to effectively reduce tissue attack during inflammatory response and it is currently in clinical phase II and III trial for the treatment of retinal neurodegenerative diseases including retinal pigmentosa (RP), caused by the loss of retinal photoreceptor cells (Sieving et al. 2006). CNTF has been found to effectively retard retinal degeneration in several animal models including rhodopsin knockout mice (Liang et al. 2001) and Q334ter rhodopsin transgenic mice (LaVail et al. 1998). The formulation of CNTF in the clinical phase I trial was delivered by retinal pigment epithelium cells that are transfected with the human CNTF gene and encapsulated in a capsule that is surgically placed in the vitreous (Sieving et al. 2006). The outer layer of the implant is permeable to CNTF and allows it to secrete from the implant once the encapsulated cells have produced the protein. Two cell lines (NTC-201-10 and NTC-201-6A) were used in the trial and they released 250 and 800 ng protein per one million cells in vitro, respectively. Three out of seven individuals showed an increase of 10–15 letters after 2 months of injection and their visual acuity was maintained for 6 months. In vivo, release rates of CNTF from the implant device remained constant for both the low and high output implant devices at 0.287 ± 0.7 and 1.53 ± 0.54 ng/day, respectively.