Potential Ocular Drug-delivery Systems for the Posterior Segment

JENNIFER J. KANG DERWENT, PhD · WILLIAM F. MIELER, MD

Since the Food and Drug Administration's approval of ranibizumab (Lucentis, Genentech) for the treatment of exudative age-related macular degeneration and increasing employment of bevacizumab (Avastin, Genentech) for various retinal vascular diseases, there is a clear need and a desire for a better means of drug delivery to the posterior segment. The current method of repeated intravitreal injections has a significant effect not only on the patients and their family members, but also on retina practices. A better treatment regimen can be accomplished by one of several means: development of more effective monotherapy treatment, combination therapy treatments, or enhanced drug delivery to the posterior segment. This article will highlight some of the potential drug delivery systems and focus on an application of thermo-responsive hydrogel as a possible platform to deliver anti-vascular endothelial growth factor (VEGF) agents.

INSERTS AND IMPLANTS

There are several potential drug delivery systems that are currently under various developmental stages and/or usage. These include solid implants such as Vitrasert, Retisert, Posurdex, Iluvien (formerly Medidur), and I-vation, to name a few. These solid devices (biodegradable and/or nonbiodegradable) are either implanted via a small surgical procedure or injected into the vitreous cavity. Devices such as Vitrasert and Retisert can release corticosteroids for an extended period of time — potentially for up to 30 months.^1-3 However, at the present time, there are no solid implants available to release anti-VEGF agents.

Biodegradable microspheres and nanoparticles offer another means of delivery of an agent to the posterior segment. Several groups are investigating the encapsulation of either pegaptanib sodium (Macugen) or Avastin into these particles for extended delivery.^4-6 These spheres/particles are usually biodegradable and are intended to be injected into the vitreous via small-gauge needles for release of the encapsulated agents. They are relatively easy to synthesize, though their effectiveness may depend on the sizes of the particles. Larger particles may scatter light and interfere with vision, while sedimentation may also occur in association with the injection and lead to localized retinal toxicity. Though there are no commercially available microspheres or nanoparticles for ocular drug delivery at the present time, these are exciting delivery platforms that need to be further investigated for potential clinical use.

Another potential delivery system that is under development is encapsulated cell technology (ECT). The technology involves use of genetically modified cells to produce ciliary neurotrophic factor (CNTF) that are encapsulated in a semipermeable, hollow fiber membrane.⁷ The device is then implanted into the vitreous and CNTF can be released for an extend period of time. CNTF released from the ECT device has been shown to be effective in an animal model. A phase 1 clinical study in humans has shown the device to be safe when implanted (Figure 1), and further studies are ongoing.

Figure 1. Histology in longitudinal section of a CNTF device after removal at 6 months from the study eye of a phase 1B participant. Histology of a comparable device that was not implanted is shown for comparison. Cells are evident at approximately equal density on the poly(ethylene terephthalate) yarn scaffold. No macrophages were found in any explanted device. Shown are 4 μm-thick sections embedded in glycol methacrylate and stained with hematoxylin and eosin (magnification ×10).

HYDROGELS

Hydrogels are also being investigated as a means of delivering anti-VEGF agents for an extended period of time. Recently, our research team has shown that thermo-responsive hydrogels can encapsulate and release active proteins, including Avastin. Thermo-responsive hydrogels have been proposed for a number of biomedical uses, including encapsulation of therapeutic agents for sustained delivery. These materials change from a solution to a gel in response to temperature change, without any additional external stimulus.

Generally speaking, the hydrogels proposed for use in biomedical applications are liquid at ambient temperatures and gel at body temperature. The novelty of the thermo-responsive hydrogel is that it can be injected through a needle or cannula, can retain the therapeutic agent in a gel state (ie, keep the agent localized), and can release it over time.

For a thermo-responsive polymer to act as an effective vehicle for drug delivery, it would have to meet a number of criteria. It would need to be capable of delivery via a relatively noninvasive method, such as a small-gauge needle, into the juxtascleral space or into the vitreous cavity. It would have to be highly efficient at encapsulating an anti-VEGF agent and then releasing it in a controlled fashion and in its active form. It would also need to be capable of sustained release of its agent for at least 3 to 6 months in order to provide a significant advantage over current delivery methods. Finally, it needs to be nontoxic, and it should be easy to manufacture, store, and use.

Poly(N-isopropylacrylamide) (PNIPAAm) is one of the polymers that exhibits thermo-responsive characteristics.⁸ At room temperature, it is a hydrated polymer that is hydrophilic in nature. With increase in temperature, it collapses into a gel and becomes hydrophobic. This phase change can occur rapidly in response to temperature change, in some cases within a minute as shown in Figure 2.

Figure 2. (Left) Appearance of the thermogel at ambient room temperature. Note the clear liquid form of the gel. (Right) Appearance of the partially solidified gel after rapid heating. Generally, the gel remains optically clear, though it was heated quickly to photographically show the transition state. In this scenario, the gel is partially opaque.

CROSS-LINKING

In order to be able to control the release of the anti-VEGF agent, the PNIPAAm was cross-linked with another polymer, poly(ethylene glycol) diacrylate (PEG-DA). This allows control of the diffusion rate through regulation of the pore size of the polymer system. Addition of PEG-DA does not greatly change the thermo-responsive nature of the PNIPAAm. The lower critical solution temperature was shifted approximately 1°C; however, this small change did not affect the delivery system.⁹

Recently, we studied the protein-release characteristics of the thermo-responsive hydrogel and subsequently measured the bioactivity of released proteins. First, thermo-responsive hydrogels were synthesized using PNIPAAm cross-linked with PEG-DA. Bovine serum albumin (BSA), and immunoglobulin G (IgG) were encapsulated into the hydrogels. With a molecular weight of 66 kDa, BSA is comparable in size to ranibizumab. IgG has a molecular weight of 150 kDa, comparable to bevacizumab. The proteins were encapsulated in hydrogels with a variety of pore sizes, as determined by the density of cross-linking, and the effect of cross-linker density on protein release was measured over time.

The cross-linked hydrogel demonstrated fast, reversible phase change when the temperature was altered. In hydrogels with lower cross-linked density, and therefore with larger pores, protein release was faster. These lower-density gels were also more pliable, meaning that the gel could be delivered through a 27-gauge needle for intravitreal delivery. Release profiles were similar for BSA and IgG, with slower release for more tightly cross-linked PEG-DA. There was an early burst of release in the first several hours, followed by the achievement of a steady state that was sustained for at least 3 weeks (duration of initial experiments). Examination of the gels after the experiment showed that a significant amount of entrapped protein remained (approximately 40%). It is anticipated that the release profiles of bevacizumab and ranibizumab will be similar to that of BSA and IgG, and this is currently being investigated.

We also investigated the potential toxic effects of the hydrogels, particularly effects due to unreacted synthesis material. There are studies demonstrating that monomers of NIPAAm can be potentially neurotoxic, though our polymerized NIPAAN was shown to be safe.¹⁰ Any unreacted synthesis material was removed by washing the hydrogels with buffer.¹¹ After polymerization, we washed the hydrogels 5 times with a large volume of sterile buffer. Each wash, samples were added to the in vitro cell culture system and measured for toxicity. After 2 washes of hydrogels, there was no toxic effect in the cell-culture system.

We also tested the safety of the thermo-responsive hydrogels in a rodent model.^12-13 The effects of intravitreal injection of the thermo-responsive hydrogels in rats were evaluated by electroretinogram (ERG) and retinal blood flow measurements. There was a small transient change in ERG and diameter of blood vessels near the hydrogel 1 week post-injection, but the effect was minimal and no significant adverse effect was noted when the animals were examined at the 1-month time frame. The details of the current finding are in preparation for publications.

The bioactivity of released proteins was also assessed. Bevacizumab and ranibizumab were encapsulated in the thermo-responsive polymer as described above and released from the gel after 1 hour, 24 hours, 48 hours, and 3 days after the temperature change. Human umbilical vein endothelial cells were stimulated with VEGF and were inhibited with the released bevacizumab and ranibizumab, showing that the released proteins were biologically active.¹¹

CONCLUSION

In summary, recently developed thermo-responsive hydrogels have been shown to be able to encapsulate and release proteins, including the anti-VEGF agents bevacizumab and ranibizumab. These agents were released by the thermo-responsive hydrogel in a bioactive form. Future work will include modification of the current gel formulation to extend the release time and to make it fully biodegradable. Thermo-responsive hydrogels appear to be a promising, minimally invasive platform for sustained delivery of drugs in the posterior segment. RP

REFERENCES

1. Fialho SL, Rêgo MB, Siqueira RC, et al. Safety and pharmacokinetics of an intravitreal biodegradable implant of dexamethasone acetate in rabbit eyes. Curr Eye Res. 2006;31:525-534.
2. Yasukawa T, Ogura Y, Sakurai E, Tabata Y, Kimura H. Intraocular sustained drug delivery using implantable polymeric devices. Adv Drug Deliv Rev. 2005;57:2033-2046.
3. Yasukawa T, Ogura Y, Kimura H, Sakurai E, Tabata Y. Drug delivery from ocular implants. Expert Opin Drug Deliv. 2006;3:261-273.
4. Amrite AC, Kompella UB. Size-dependent disposition of nanoparticles and microparticles following subconjunctival administration. J Pharm Pharmacol. 2005;57:1555-1563.
5. Amrite AC, Edelhauser HF, Singh SR, Kompella UB. Effect of circulation on the disposition and ocular tissue distribution of 20 nm nanoparticles after periocular administration. Mol Vis. 280;14:150-160.
6. Sahoo SK, Dilnawaz F, Krishnakumar S. Nanotechnology in ocular drug delivery. Drug Discovery Today. 2008;13:144-151.
7. Tao W. Application of encapsulated cell technology for retinal degenerative diseases. Expert Opin Biol Ther. 2006;6:717-726.
8. Heskin M, Guillet JE. Solution properties of poly(N-isopropylacrylamide). J Macromolecular Science. 1968;2:1441-1455.
9. Kang Derwent JJ, Mieler WF. Thermo-responsive hydrogels as a new ocular drug delivery platform to the posterior segment of the eye. Trans Am Opthalmol Soc. 2008;106:206-213.
10. Hashimoto K, Sakamoto J, Tanji H. Neurotoxicity of arcylamide and related compounds and their effects on male gonads in mice. Arch Toxicol. 1981;47:179-189.
11. Kang Derwent JJ, Mieler WF. Drug delivery to the posterior segment via thermo-responsive hydrogels. Paper presented at: Retina Society 41st Annual Scientific Meeting; Scottsdale, AZ; September 25-28, 2008.
12. Guthrie M, Appel A, Benac S, et al. Electroretinographic assessment of retinal function in response to intravitreal injection of thermo-responsive hydrogel drug delivery system. Invest Ophthalmol Vis Sci. 2008;50:E-abstract 5997.
13. Kang Derwent JJ, Appel A, Benac S, et al. Biocompatibility of thermo-responsive hydrogel ocular drug delivery system in rodent model. Invest Ophthalmol Vis Sci. 2008;50:E-abstract 6000.