14C-mannitol into albino rabbits (Lee and Robinson 2001). After 50 mL of 1.82 mM 14C-mannitol subconjunctival injection, the vitreous and aqueous drug concentrations were 18.7 and 261 nM, respectively, after 0.5 h postadministration. However, when 10 mL of 1.82 mM 14C-mannitol was injected intracamerally, the vitreous and aqueous drug concentrations were 0.348 mM and 2.57 nM, respectively, after 0.5 h. At 5 times the concentration of the intracameral injected dose, the subconjunctival dose had 133 times less mannitol concentration in the aqueous humor and about 7.3 times higher concentration of mannitol in the vitreous humor. Thus, the ratio of aqueous to vitreous humor drug level was much higher for intracameral injections as compared to subconjunctival injections (13,600 vs. 14). Therefore, while intracameral route is efficient in distributing hydrophilic macromolecules to the aqueous humor, subconjunctival route is more efficient in the relative distribution of drugs to the vitreous humor of the eye.

Interestingly, a separate study reported that intraocular injections (both intravitreal and intracameral) of bevacizumab were more effective in treating corneal ­neovascularization than subconjunctival injections in mice (Dratviman-Storobinsky et al. 2009). Mice were administered 25 mg/mL of intracameral, intravitreal, or subconjunctival injections of bevacizumab and the neovascularization areas were measured. The relative areas of corneal neovascularization after 10 days were: 19.86 ± 1.23, 24.20 ± 14.87, and 39.73 ± 14.51% for intracameral, intravitreal, and subconjunctival administrations, respectively, compared to the untreated mice (50.62 ± 24.74%). Similarly on day two, four, and eight, a similar trend was seen. That is, intracameral and intravitreal administrations resulted in lower corneal ­neovascularization areas as compared to subconjunctival route. These differences might be due to more rapid protein clearance from the subconjunctival space as opposed to slower clearance from the vitreous and due to exposure of high peak concentrations to the cornea following intracameral injections. However, all three administrations had lower corneal neovascularization areas as compared to untreated group. Thus, in the treatment of anterior ocular illnesses such as corneal neovascularization, intracameral injections may be more appropriate than subconjunctival. However, the focus of this chapter is on protein delivery to the posterior segment of the eye and thus far, intracameral injection of protein therapeutics has not been ­successful in delivering therapeutic quantities to the posterior segment of the eye.

17.2.3  Intravitreal

In 1940, intravitreal injections of penicillin were first injected into the posterior ­segment of the affected eye in combination with sulfadiazine to treat ocular ­infections of P. aeruginosa (Spencer 1953; Baum et al. 1982). Since then, off-label intravitreal injections have found a wide range of clinical applications for the treatment of various diseases of the posterior segment of the eye. Macromolecules are relatively well suited for this route of administration as they are retained for a long period of time within the vitreous cavity after intravitreal injection, and this leads to an increase in the duration of

pharmacological action. Within the vitreous, high molecular weight species were found to have a much longer half-life than low molecular weight species­ (Durairaj et al. 2009). Intravitreal injections are desirable as they inject the drug directly into the vicinity of the targeted tissue (i.e., the posterior segment of the eye, which is difficult to reach by other routes of administration) and in addition, they are relatively safe, although several rare complications have been observed. After a single intravitreal injection of 1.25 mg (0.05 mL) of bevacizumab administered to rabbit eyes, its concentration was maintained in the vitreous at >10 mg/mL for 30 days. The half-life of bevacizumab in the vitreous was reported to be 4.32 days in rabbits with a peak concentration of 37.7 mg/mL in the aqueous humor after only 3 days of administration (Bakri et al. 2007b). However, in serum, the maximum concentration of bevacizumab was 3.3 mg/mL after 8 days and fell below 1 mg/mL after 29 days of injection. In the untreated contralateral eye, negligible amount of drug was found (0.35 ng/mL after day one and 11.17 ng/mL after 4 weeks). This route of administration provides high concentrations of therapeutic agents within the proximity of target tissues of the back of the eye. Therefore, for high efficacy in treating retinal disorders, the intravitreal route of administration is the preferred route for macromolecules. However, repeated intravitreal injections can result in various complications including cataracts, retinal detachment, and endophthalmitis (Raghava et al. 2004). In addition, depending on the location of the target tissue, macromolecules may experience difficulty in permeating cellular membranes as well as intercellular junctions due to their extremely large molecular size and surface charge.

17.2.4  Periocular (Transscleral)

A potential solution to the many safety limitations of intravitreal injections is to use a transscleral delivery system, which is less invasive to the globe and hence it might reduce the incidence of cataract, retinal detachment, and endophthalmitis. The transscleral route includes periocular routes of delivery such as subconjuntival, sub-Tenon, posterior juxtascleral, and retrobulbular injections (Olsen et al. 1995; Ambati et al. 2000; Raghava et al. 2004). Depending on the site of action, the drug may have to cross the sclera, choroid, and/or the RPE to exert its effects in the posterior ­segment of the eye. Since drugs are much more permeable across the sclera than the cornea (Prausnitz and Noonan 1998), many investigators have assessed the periocular route of administration. Although high molecular weight compounds up to 150 kDa (fluorescein isothiocyanate-rabbit immunoglubulin) are able to permeate the sclera with a permeability coefficient of 1.34 ± 0.88 × 10−6 cm/s, this value is much lower compared to 84.5 ± 16.1 × 10−6 cm/s observed for a 376-Da (sodium fluorescein) compound (Ambati et al. 2000). Although small molecules as well as macromolecules permeate across the sclera, the permeability decreases with an increase in size (Prausnitz and Noonan 1998; Ambati et al. 2000).

Periocular injections of an adenoviral vector expressing either FLT-1.10 (soluble VEGF receptor 1) or TGF-b.10 (transforming growth factor beta) or PEDF.11 ­(pigment epithelium derived factor) showed similar concentration levels in both the

detachment, it is an excellent route for delivering drug to the retina while escaping most of the delivery barriers such as membrane and enzymatic degradation barriers (Steele et al. 1993).

Goat immunoglobulin (goat-IgG) adsorbed on gold nanoparticles was successfully delivered to photoreceptor cells as well as RPE when injected subretinally (Hayashi et al. 2009). Rabbit sclera was punctured using a 25-gauge needle and a subretinal cannula was inserted. Goat-IgG (0.15–0.2 mL) either adsorbed on nanoparticles and suspended in PBS or directly dissolved in PBS was injected into the subretinal space using a 28-gauge needle. Nanoparticles successfully delivered the goat-IgG locally to photoreceptor cells as well as RPE as confirmed by immunohistochemistry and transmission electron microscopy; however, 1 week after injection, there was extensive retinal degeneration. It was argued that the retinal degeneration was specific to rabbits because rabbit retina is mostly avascular, in which case the retina largely depends upon the choroid for its nourishment. One of the major side effects of subretinal injection is temporary retinal detachment, which may in fact initiate retinal degeneration. The same may not be true for species with extensive retinal blood supply and hence this route may still be promising to deliver drug locally to the retina.

A novel cell penetrating peptide for ocular delivery (POD) was found to be an effective delivery system for delivering green fluorescent protein (GFP) to the ­photoreceptor and RPE cells via subretinal injections (Steele et al. 1993). Approximately 8.5 mg of POD-GFP was injected in the subretinal space of mice and the eyes were enucleated after 6 h. The presence of POD-GFP was examined by immunocytochemistry. POD-GFP was detected in both photoreceptor cells and in RPE cells. Electroretinography (ERG) showed no significant toxicity. This route of administration may be promising for delivering proteins and macromolecules, but great care has to be taken in the injection procedure to prevent complications associated with the injection technique such as retinal detachment and degeneration.

17.2.7  Systemic

Systemic route involves injection of subcutaneously, intramuscularly, intraperitoneally, or intravenously. Although this route is safe and has better patient compliance in comparison to intravitreal or other routes of administration, this route fails to deliver therapeutic concentrations of drug in efficient­ manner to the back of the eye due to the presence of blood-retinal and enzymatic barriers. Protein or other therapeutic agents delivered via systemic route reach the back of the eye in very limited quantity (Furrer et al. 2009). After systemic administration of radiolabeled chimeric protein IL-2 and pseudomonas exotoxin PE40, a very high level of drug was detected in blood, liver, and spleen; however, there was an undetectable quantity of drug in ocular tissues (BenEzra et al. 1995). Systemic delivery of bevacizumab for neovascular AMD was evaluated in an uncontrolled clinical study SANA [systemic bevacizumab (Avastin) therapy for neovascular age-related macular