Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

Glossary

Bioengineered corneas – Are natural-based substitutes for human donor tissue that are designed to replace part or the full thickness of damaged or diseased corneas.

Collagen – The most common naturally occurring structural protein found in all multi-cellular animals and accounts for approximately 30% of all body proteins.

Interpenetrating polymeric networks (IPNs) –

Polymeric structures comprising two or more networks that are interconnected on a molecular scale through chemical (covalent) and physical bonds.

Keratoprosthesis – A type of artificial cornea that is designed to be implanted in a patient who has severe bilateral corneal disease for which a corneal transplant is not an option.

Lenticles – A tiny disk slipped into the pocket of the patient’s own cornea between corneal epithelium and Bowman’s membrane for vision correction.

Penetrating keratoplasty – A surgical procedure where a damaged or diseased cornea is replaced by donated corneal tissue which has been removed from a recently deceased individual having no known diseases which might affect the viability of the donated tissue.

Photorefractive keratectomy (PRK), laser-assisted sub-epithelial keratectomy (LASEK), or laser-assisted in situ keratomileusis (LASIK) – Laser eye-surgery procedures for correcting a person’s vision can reduce the need for glasses or contact lenses.

The Need for Artificial Corneas

The terms artificial corneas (ACs) or bioengineered corneas are widely used to describe corneal scaffolds that are designed to restore vision. These scaffolds are used as substitutes to human donor corneas, and can replace part or the full thickness of damaged or diseased corneas.

Corneal scaffolds can range from solely synthetic ocular prostheses through to tissue-engineered hydrogels that allow some regeneration of the host tissues. In addition, bioengineered lenticles may be implanted into the cornea to improve vision. This is achieved by altering the refractive properties of the eye, which is an alternative procedure to laser-assisted in-situ keratomileusis (LASIK), laser-assisted sub-epithelial keratectomy (LASEK), and photorefractive keratectomy (PRK).

Compromising the transparency of the cornea interferes with its function. Once optical clarity is compromised, due to disease or damage, vision loss occurs and can result in corneal blindness (Figure 1). According to the World Health Organization (WHO), there are approximately 37 million people worldwide who possess bilateral blindness, as well as at least 124 million people who have impaired vision in both eyes. Corneal ulceration and ocular trauma are reported to be the major causes of corneal blindness, accounting for 1.5–2 million new cases annually. At present, transplantation of matched human donor tissue is the only widely acceptable treatment.

Corneas are the most successful organ transplants – with an 86% graft-survival rate at the 1-year postoperative follow-up, 73% after a 5-year period, 62% after 10 years, and 55% at 15 years. The success of the transplantation is dependent upon the availability of goodquality donor tissue, as well as the patient’s condition. Inactive central scars or keratoconus are amenable to transplantation; however, alkali burns or neurotrophic scars that are secondary to Herpes zoster ophthalmicus have a poor prognosis. The cornea donor pool is in limited supply due to a longer life expectancy, combined with the aging population within North America. Demand for corneas is expected to increase, but a shortage in supply will most likely be experienced. The shortage is expected to be compounded by the increasing incidence of infectious diseases (HIV, hepatitis, Creutzfeldt–Jakob disease, etc.), as well as the growing popularity of refractive surgery. Surgically treated corneas are thinned, rendering them unacceptable as donor tissue. As alternatives to donor tissues, bioengineered corneas are designed to replace some or all of a damaged or diseased cornea. They range from prosthetic devices that solely address replacement of the cornea’s function, to tissue-engineered hydrogels that permit regeneration of host tissues. This article focuses on the efforts employed to build an in vitro model of the visual system.

In particular, we examine the development of novel biomaterials that serve as the building blocks for the fabrication of scaffolds in engineered tissues.

Desired Characteristics for an

Implantable AC

In order to be clinically applicable, fabricated corneal substitutes need to replicate the functions of the human cornea. The human cornea forms a transparent window through which light is transmitted to the retina, enabling one to see. A unique property of the cornea is its optical clarity, which accounts for over 70% of the light that is transmitted to the retina for vision. Corneal clarity is now believed to result from a combination of refractive-index matching, and the presence of structural components that are well below the wavelength of visible light.

As alternatives to donor tissues, ACs are designed to replace some or all of a damaged or diseased cornea. They range from prosthetic devices that solely address replacement of the cornea’s function, to tissue-engineered hydrogels that permit regeneration of host tissues. In instances where corneal stem cells have been depleted by injury or disease, tissue-engineered lamellar implants reconstructed with stem cells have been transplanted. In situ methods using ultraviolet A (UVA) cross-linking have also been developed to strengthen weakened corneas. In addition to the clinical need, bioengineered corneas are also rapidly gaining importance in the area of in vitro toxicology. In Europe, there is

currently a ban on consumer product testing in animals (European Union Directive 76/768/EEC) that is expected to expand worldwide. Complex, fully innervated, physiologically active, three-dimensional (3D) organotypic corneal models are currently being developed and tested.

ACs must meet certain requirements, without exception, to be successful. To properly integrate into host tissue, AC must be biocompatible and noncytotoxic. Awatertight junction with the host tissue is essential for preventing infection and epithelial down-growth. Epithelial cell growth must be supported over the anterior surface, allowing a wettable, self-renewing layer that promotes a healthy tear-film formation. Nerve innervations must be supported for high touch sensitivity. Penetration and proliferation of host fibroblast cells must be promoted for tissue regeneration. Optical transparency >80% and light scatter of <5% should be exhibited, as well as a suitable morphology and curvature to obtain the appropriate refractive index. In addition, flexibility and sufficient tensile strength is required to allow surgical manipulation and fixation, as well as to protect the eye. The AC should exhibit sufficient swelling in aqueous solutions similar to that of native cornea, but at the same time be permeable to oxygen, nutrients such as glucose, and serum albumin – which is a major water-soluble protein in the human cornea. Lastly, the AC must be inexpensive and easy to fabricate.

Artificial or bioengineered corneas developed to date range from prostheses – known as keratoprosthesis (KPro) – to naturally fabricated cell-based tissue equivalents, to bioengineered scaffolds that serve as templates for regeneration of host tissues.

Synthetic Artificial Corneas or

Keratoprosthesis

Research into the development of an AC has existed for more than 200 years, with the original glass and quartz optics being put forward, in 1789, by Guillaume Pellier de Quengsy. Since then, there have been numerous attempts at developing ACs. Currently, four generations of KPros have been defined, according to the KPro Study group. KPros are synthetic implants designed to replace the central portion of an opaque cornea. First-generation KPros are comprised of monoblocks, or one-piece prostheses that are made from plastics such as poly methyl methacrylate (PMMA). An example of a second-generation KPros is the osteo-odonto keratoprosthesis (OOKP). This prosthesis, developed by Strampelli in 1964, consists of autologous tissue derived from tooth and bone, which surrounds a central PMMA optic. An osteodental skirt is preimplanted into the buccal mucosa, allowing colonization of fibroblasts to support its integration as an ocular implant. Third-generation KPros include a range of devices with plastic optics and metal parts that aid in anchoring the device to host tissues. Donor-tissue attachment can also be achieved through skirts, allowing for host integration. Fourth-generation KPros utilize the optic-skirt model, in which a solid optical core is surrounded by a porous skirt. This encourages biointegration with the adjacent host tissues to circumvent implant extrusion. Rigid synthetic polymers, such as PMMA, were used in the early attempts to develop artificial corneal transplants. While PMMA still remains a popular material, poly(2-hydroxyethyl methacrylate) (PHEMA) has been used more frequently in various types of KPros. In this article, we provide a synopsis of several examples of KPros that have either been tested clinically, or are currently in clinical use.

KPros currently in clinical use include the following: the OCULAIDW KPro, Dohlman KPro, AlphaCor™Kpro, OOKP KPro, BioKPro III, Seoul-type KPro, and Pintucci KPro. Figure 2(a) and 2(b) represents the OCULAIDW KPro, composed of an anti-conical shaped shaft that can be fixed into the host cornea or sclera. It creates a valve on the cornea to ensure a watertight environment. The pressure in the eye pushes the corneal rim around the 3-mm top of the KPro, while the steel-suture fixation on the sclera is designed to prevent extrusion. The Dohlman AC is a collar- button-design KPro, composed of PMMA. It consists of a central optical stem that penetrates the full thickness of the cornea. This stem is sandwiched between two plates and sutured into place similar to a penetrating keratoplasty (PKP) graft (Figure 2(c)).

The AlphaCor™ Kpro is one of the best-known keratoprosthetic devices fabricated as a one-piece device that comprises a transparent core and an opaque porous skirt. The implant is a 7-mm-diameter, one-piece, nonrigid synthetic cornea (Figure 2(d)). Composed of a transparent

central optic core of PHEMA gel, the KPro is designed to allow the passage of light into the posterior of the eye. The outer porous skirt – an opaque, high-water PHEMA – is designed to allow cell infiltration from the host, which anchors the prosthesis into place. This device gained the Food and Drug Administration (FDA)-approval in 2002 for use in patients with scarred, vascularized, or diseased corneal tissues who are either ineligible for conventional donor-tissue transplants or have had multiple previous graft failures. Early results suggest that the AlphaCor™ Kpro, previously known as the Chirila KPro, is clinically safe. Associated complications include the formation of retroprosthetic membranes, corneal melt, retained lenticular material, optic depositions, and rare cases of device extrusion. Contraindications include abnormal tear film, as well as uncontrollable high intraocular pressure. Topical administration of medroxyprogesterone has been shown to limit corneal melting of the device. However, this KPro has been effectively used to restore a degree of vision in patients considered untreatable by conventional corneal transplantation.

The OOKP, described above, consists of autologous tissue derived from tooth, which surrounds a central PMMA optic. This KPro is one of the most successful, as it has a low extrusion rate due to the excellent integration of the skirt material with the host tissue. Associated complications with the OOKP include retroprosthetic membrane formation, glaucoma and decentration of the central optic, due to absorption of the osteodental skirt.

The BioKPro III, designed by the Legeais group, was recently clinically evaluated at the Moorfields Eye Hospital, London, UK. The BioKpro III consists of a central 5-mm-diameter, 500-mm-thick silicone optic, and a surrounding opaque skirt made of porous fluorocarbon. The device was implanted into seven patients with severe corneal scarring, and monitoring occurred between 18 and 48 months. The results showed that the KPro failed in six patients, due to extrusion occurring between 2 and 28 months postoperatively. One patient, who had a thermal burn, retained the KPro and reported an improvement in vision. However, this patient also reported mucous accumulation on the optic.

The Seoul-type KPro (S-KPro) consists of three sections: a long cylindrical optic surrounded by a mushroomshaped anterior flange, a skirt for corneal fixation, and haptics for scleral fixation. The 4-mm diameter optic is made of PMMA; the anterior flange is composed of fluorinated silicone approximately 0.2-mm thick and 6 mm in diameter; while the skirt is fabricated from expanded polytetrafluoroethylene (e-PTFE). Preliminary results from the first human trial indicate that complications including retinal detachment, formation of a retroprosthetic membrane, and extrusion have been identified.

The Pintucci KPro consists of a 3-mm thick and 5-mm long optical cylinder made of PMMA. A woven, 0.7-mm

thick and 10-mm circular Dacron membrane is added to the KPro for tissue integration. Like the OOKP, this device is preimplanted into the patient for colonization of the skirt. Thirty-one patients have received implants from 1997 to 2004, by means of clinical trials. Results indicated that no infections or retroprosthetic membranes were reported. Seventy-seven percent of implanted eyes improved enough to enable the patients to function independently. However, approximately 40% of implanted eyes had complications, although only a few cases were vision threatening.

Recent developments in the design of KPros include a KPro developed by Storsberg and colleagues in Germany; the Stanford KPro; and recent work by Sheardown and colleagues on the coverage of KPros by extracellular matrix (ECM) proteins for enhanced epithelialization. The German KPro adheres to the eye without sutures, reducing inflammation and infection. Sheardown and colleagues have covalently attached cell-adhesion peptides to poly(dimethyl siloxane) (PDMS) surfaces. This surface modification has been effective, leading to a synergistic effect on corneal epithelial cell attachment when compared to single peptides only. The Stanford KPro has also been designed using a polymer network hydrogel, comprised of poly(ethylene glycol) and poly(acrylic acid) (PEG/PAA). When implanted in rabbit corneas, this hydrogel was retained and tolerated well in nine out of 10 cases for a 2-week period.

To date, however, no KPro meets the previously defined standards for a successful corneal implant. As such, no particular KPro is in widespread use to date, although recent versions appear to be promising.

Self-Assembled Corneal Equivalents

There have been various attempts at developing a naturalbased self-assembled corneal equivalent. These range from the use of purely biological materials synthesized by cells in culture, to the use of noncorneal tissues as substitutes. Most bioengineering approaches to the restoration and repair of damaged tissue require scaffold materials upon which cells can attach, proliferate, and differentiate. Such scaffolds can be made using a self-assembly approach in which chemicals are used to stimulate the secretion of collagen, and other ECM molecules by fibroblast cells. Resulting sheets of scaffolds are stacked together to form a stroma, allowed to further integrate in vitro, and then epithelial cells are seeded on top of the stack. These constructs mimic corneal morphology, and the cells express appropriate tissue-specific markers. The main drawback is the time needed to produce enough self-assembled scaffolding for transplantation.

A cornea equivalent, composed of a 3D, bovine, dermal collagen matrix has been developed by Minami and

colleagues for in vitro studies. Zieske and colleagues also developed an in vitro cornea – fabricated using primary rabbit stromal cells. Funderburgh and colleagues have used keratocytes of the corneal stroma to produce a transparent ECM that may be useful in cell-based corneal therapy or for the development of bioengineered corneas. Funderburgh and colleagues also determined there is a population of cells present in adult mammalian corneal stroma having the ability to divide extensively, generating differentiated keratocytes. Previously, the authors reconstructed a human cornea using immortalized human corneal cell lines. Each cell line was subjected to electrophysiological, biochemical, and morphological tests. This was carried out to determine the phenotype, which was compared to postmortem human corneal cells, before being used in the 3D reconstruction. Collagen–chondroitin sulfate was the base scaffold in which keratocytes were integrated, before epithelial and endothelial cells were layered above or below. Two weeks following construction, the resulting corneal equivalent was found to behave similarly to a normal cornea, with respect to morphology, transparency, ion and fluid transport, and gene expression following injury. Although this human corneal equivalent shared functional properties with the natural cornea, it was not designed to meet the mechanical characteristics needed for transplantation. However, these studies represent an important future directive toward the development of bioengineered corneal implants. Reconstructed corneal equivalents presently have use in the biomedical world, as they are used as replacements for animals in toxicology testing and pharmacological studies.

Bioengineered ACs that Address Regeneration

To overcome the challenges of biocompatibility, inflammatory responses, and rejection, there have been attempts to promote varying degrees of corneal tissue regeneration through implants of bioengineered corneal ECM substitutes. In general, these matrix-mimetic materials range from simple cross-linked ECM macromolecules, such as collagen, to hybrids of ECM macromolecules and synthetic polymeric components. Prior to the assembly of these ECM mimetics for implantation, a bioactive scaffold material with accurate chemical/physical properties must be designed. It must be able to form robust scaffolds, promote cell differentiation/integration, and promote tissue formation in a uniform manner that is repeatable and reliable.

Polymeric blends of collagen have been previously used to emulate the collagen–glycosaminoglycans scaffolding of the ECM. Various tissue-engineering applications have utilized this technology, for example, as a scaffold for artificial liver, skin scaffolds with nerves and dermal models, membranes for controlled drug release, and as an in vitro model to test antineoplastic agents. Many

efforts have been made to stabilize collagen, and its blends, by chemical cross-linking methods. These methods can be divided into two categories: bifunctional and amide-type. Several bi-functional reagents such as glutaraldehyde (GTA), polyethylene glycol diacrylate (PEGDA), and hexamethylene diisocyanate (HDC) have been used to bridge amine groups of lysine or hydroxylysine residues of collagen polypeptide chains. A major handicap of these cross-linking agents is the potential toxic effect of residual molecules and/or compounds released when the biomaterial is exposed to biological environments (i.e., during in vivo degradation).

Amide-type cross-linkers such as carbodiimide, especially 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS) offer the main advantage of lower toxicity and better compatibility over GTA and HDC. However, collagen scaffolds stabilized by carbodiimide are not strong, yet are elastic enough for PKP transplantation. This is due to limited zero-length crosslinks; in addition, there are reaction sites on collagen molecules that are not linkable by carbodiimide. It is reported that EDC and NHS (EDC/NHS) can link carboxylic acid and amino groups located within 1 nm from each other. Therefore, functional groups that are located on adjacent collagen microfibrils are too far apart to be bridged by carbodiimide. With systems such as EDC/NHS, the increase in tensile strength, especially when induced by the increasing of a cross-linking agent, is associated with the decrease in elasticity and toughness. This compromise in integrity may be due to restraints placed on the mobility of the polymer network, a decrease in scaffold porosity, and/or a diffusion of reactive residues and byproducts out of the scaffolds.

Despite the drawbacks, cross-linking agents have been used effectively to fabricate collagen based matrices. The application of these matrices has been mainly diagnostic; where the tissue is made and used in vitro for testing drug metabolism, uptake, and toxicity.

The authors have previously reported collagen-based materials, ranging from corneal scaffolds based on the copolymer poly(N-isopropylacrylamide-co-acrylicacid-co- acryloxylsuccinimide), to a simple EDC/NHS cross-linked collagen scaffold. These scaffolds allow regeneration of corneal cells and nerves, when implanted as lamellar grafts. However, these materials still lack the optimal toughness and elasticity required to withstand PKP surgical procedures, as well as normal day-to-day mechanical stresses.

In our most recent ongoing clinical study, we reported corneal regeneration following the implantation of a bioengineered corneal substitute, based on recombinant collagen. Visual acuity, ocular surface quality, and corneal sensitivity are continuously improving in the first recipients of the implants. Figure 3 shows slit-lamp and optical coherence tomography (OCT) photographs of the operated cornea immediately following lamellar keratoplasty and at 9 months post operation. Such substitutes

may find use as temporary or emergency corneal replacements, where human tissue is unavailable.

In several attempts to enhance mechanical properties of corneal substitutes, hybrid interpenetrating polymeric networks (IPNs) have been developed as ACs. Synthetic-based IPNs have been widely explored and used; however, bioengineered IPNs have not been investigated and examined for corneal applications until recently. Efforts were made by the authors to develop a bioengineered IPN as an AC. The goal was to develop an IPN scaffold that combines the bioactive features of biopolymers with the physical characteristics of synthetic polymers. Composite IPN structures – comprised of two or more interconnected networks on a molecular scale through chemical (covalent) and physical bonds – were developed.

The scaffold material used bio-functional polymers that naturally occur in the native tissue (e.g., collagen) as the core material. As an alternative, tissue-mimetic polymers such as chitosan, 2-methacryloyloxy ethyl phosphorylocholine (MPC) or chondroitin sulfate were used as bio-interactive components; with poly (ethylene glycol) dibutyraldehyde (PEG-DBA) or poly(ethylene glycol) diacrylate (PEG-DA) being used as a synthetic long-range cross-linker. Bio-inert short-range cross-linkers, including EDC and NHS, were also used in conjunction with long-range cross-linkers. This composition allowed the formation of hybrid IPNs that are mimetic of the natural cornea. The IPN scaffolds demonstrated significantly enhanced mechanical strength and elasticity compared to

12 mm

0.5 mm

~3 mm

(c)

Figure 4 Corneal implants: (a) bioengineered artificial cornea (IPN), (b) eye-bank human donor cornea, and (c) dimensions of a typical bioengineered artificial cornea.

their non-IPN counterpart. In addition, they demonstrated excellent optical properties, optimum mechanical properties and suturability, and good diffusivity to glucose and albumin. The IPNs had excellent biocompatibility, and were further tested by being implanted into pig corneas. Over the 12-month monitoring period, it was demonstrated that there was seamless host–graft integration with successful regeneration of host corneal epithelium, stroma, and nerves. Figure 4 depicts an IPN-bioengineered AC compared to an eye-bank human donor cornea.

Future Directions

In the cornea, regeneration of the host cornea could overcome the rejection problems and other postoperative complications from donor-tissue transplantation and KPros. In addition, corneal implants that allow nerveand host-tissue regeneration could also circumvent problems after surgery, found both in human donor tissue and in synthetic KPros. At the current pace of product development and testing, viable alternatives to donor corneas for transplantation are not far off. Processing procedures can affect both the native material properties and the subsequent clinical utility of scaffolds intended for certain tissue-engineering applications. Irrespective of the method, the AC must be engineered to mimic morphological, physiological, and biochemical properties of the natural tissue as closely as possible.

See also: Corneal Epithelium: Cell Biology and Basic Science; The Corneal Stroma; Cornea Overview; Penetrating Keratoplasty; Refractive Surgery and Inlays.

Further Reading

Chirila, T. V., Hicks, C. R., Dalton, P. D., et al. (1998). Artificial cornea.

Progress in Polymer Science 23: 447–473.

Crawford, G. J., Hicks, C. R., Lou, X., et al. (2002). The Chirila keratoprosthesis: Phase I human clinical trial. Ophthalmology 109: 883–889.

Dohlman, C. H., Harissi-Dagher, M., Khan, B. F., et al. (2006). Introduction to the use of the Boston keratoprosthesis. Expert Review of Ophthalmology 1(1): 41–48.

Duan, D., Klenkler, B. J., and Sheardown, H. (2006). Progress in the development of a corneal replacement: Keratoprostheses and tissue-engineered corneas. Expert Review of Medical Devices

3: 59–72.

Funderburgh, M. L., Du, Y., Mann, M. M., Raj, N. S., and Funderburgh, J. L. (2005). PAX6 expression identifies progenitor cells for corneal keratocytes. FASEB Journal 19(10): 1371–1373.

Germain, L., Carrier, P., Auger, F. A., Salesse, C., and Gue´rin, S. L. (2000). Can we produce a human corneal equivalent by tissue engineering? Progress in Retinal and Eye Research 19: 497–527.

Griffith, M., Hakim, M., Shimmura, S., et al. (2002). Artificial human corneas: Scaffolds for transplantation and host regeneration. Cornea 21(7): S54–S61.

Griffith, M., Osborne, R., Munger, R., et al. (1999). Functional human corneal equivalents constructed from cell lines. Science

286: 2169–2172.

Hicks, C., Crawford, G., Chirila, T., et al. (2000). Development and clinical assessment of an artificial cornea. Progress in Retinal and Eye Research 19: 149–170.

Kalayoglu, M. V. (2006). In search of the artificial cornea: Recent developments in keratoprostheses. Ophthalmology Technology Spotlight Medcompare.

Leibowitz, H. M., Trinkhaus-Randall, V., Tsuk, A. G., and Franzbau, C. (1994). Progress in the development of a synthetic cornea. Progress in Retinal and Eye Research 13: 605–621.

Minami, Y., Sugihara, H., and Oono, S. (1993). Reconstruction of cornea in three-dimensional collagen gel matrix. Investigative Ophthalmology and Visual Science 34: 2316–2324.

Rafat, M., Li, F., Fagerholm, P., et al. (2008). PEG-stabilized carbodiimide crosslinked collagen–chitosan hydrogels for corneal tissue engineering. Biomaterials 29: 3960–3972.

Resnikoff, S., Pascolini, D., Etya’ale, D., et al. (2004). Global data on visual impairment in the year 2002. Bulletin of the World Health Organization 82: 844–851.

The National Coalition for Vision Health (2005). There’s still a critical shortage of corneas for transplantation 2005. http://www. visionhealth.ca/news/insert/shortage.htm (accessed July 2009).

WHO (1997). Global initiative for the elimination of avoidable blindness. Geneva: World Health Organization. (unpublished document WHO/PBL/97.61/Rev 1). http://whqlibdoc.who.int/hq/1997/ WHO_PBL_97.61_Rev.1.pdf.

WHO (2003). Human organ and tissue transplantation. World Health Organization, May 2003 (EB112/5 112th Session). http://www.who. int/ethics/topics/human_transplant/en (accessed July 2009).

Worst, J. (2007). The ‘‘Champagne Cork’’ keratoprosthesis (KP). http:// www.janworst.com/projects/kp/frames/framekp1.htm.

Yaghouti, F., Nouri, M., Abad, J. C., and Power, W. J. (2001). Keratoprosthesis: Preoperative prognostic categories. Cornea 20: 19–23.

Relevant Website

http://www.asmr.org.au – The Australian Society for Medical Research, Following in the footsteps of Fred Hollow, Key Statistics 2006.

Glossary

Amidases – The enzymes that catalyze the cleavage of carbon–nitrogen bonds in amides. Bioreversion – The conversion of a prodrug to an active form.

Esterases – The enzymes that catalyze the hydrolysis of an ester into its alcohol and acid. Peptidases – The enzymes that catalyze the hydrolysis of peptides into amino acids. Prodrug – The drugs designed to be inactive until

in vivo activation generates the active form of the drug. Transporter – The protein that translocates materials in biological systems resulting from expenditure of metabolic energy.

Topical Ocular Drug Delivery

Topical drug delivery is the most acceptable form of treatment for the diseases affecting the anterior segment such as corneal epithelial and stromal keratitis, glaucoma, conjunctivitis, dry eye syndrome, iritis, uveitis, and blepharitis. The structure of the eye is shown in Figure 1. Topically applied drugs can reach the intraocular tissues either by corneal and/or noncorneal (conjunctival–scleral) pathway(s). Drugs traversing the corneal pathway should permeate through the corneal epithelium and stroma which are rate-limiting barriers for hydrophilic and lipophilic molecules, respectively. Conjunctival absorption could result in higher drug concentrations in the anterior as well posterior chambers depending on the mechanism of absorption. Conjunctival–scleral pathway is favored for the treatment of diseases in the posterior segment, as it can bypass the anterior chamber and thus permit direct access to intraocular tissues like sclera retina and vitreous humor. However, nasolacrimal drainage, tear dilution, as well as the outer conjunctiva and cornea, act as barriers to drug absorption. As a result, therapeutic concentrations in intraocular tissues following topical administration are difficult to achieve and about 1–5% of topically instilled dose reaches the anterior segment of the eye. Moreover, a larger fraction of the applied drug is eliminated from the precorneal area within 5 min, through drainage across

nasolacrimal duct into the systemic circulation. Fifty percent of the normal human tear film is replaced every 2–20 min. Such a high tear-turnover rate also reduces the drug-residence time in precorneal and conjunctival areas. Therefore, rapid tear-film drainage can also impede the drug absorption following topical administration. Topically administered agents have a low probability of reaching the posterior segment in significant amounts, as passage through the corneal and conjunctival epithelia, aqueous humor, and lens is required to reach the retina.

Various strategies have been investigated in order to improve the corneal and conjunctival absorption of drugs instilled topically. Prodrug approach is one of the most promising and effective strategies currently being investigated for ophthalmic drug delivery. Exploring the inherent drug metabolism capability of ocular tissues is one of the important aspects of prodrug design. In this strategy, the drug molecule is modified chemically by attaching it to a promoiety to improve the physicochemical characteristics, such that higher drug absorption into the tissues can be achieved.

Prodrugs are designed to be therapeutically inactive until in vivo activation to generate the parent drug. The compounds are synthesized by linking an appropriate chemical moiety to the parent drug, usually linking by an ester or an amide bond. Upon absorption into the tissue, the prodrug will be subjected to enzymatic hydrolysis (bioreversion by esterases/peptidases) to release the active parent drug (Figure 2). The rate of bioreversion depends upon various factors, including affinity of the prodrug linkage toward hydrolyzing enzyme(s), the capacity and turnover rate of the enzyme, etc. The enzymes responsible for hydrolysis of prodrugs are present ubiquitously in all biological fluids and tissues. For example, esterases are expressed throughout the body and can be utilized in the hydrolysis of an ester functional group. In ocular tissues, the esterase activity has been found to be the highest in iris-ciliary body followed by the cornea and the aqueous humor. Among ocular esterases, butyrylcholinesterase (BuChE) constitutes the major proportion compared to acetylcholinesterase (AChE) – except in the corneal epithelium of albino rabbits. Drugs and prodrugs containing ester linkages can undergo varying extents of esterase-mediated hydrolysis while permeating the cornea/conjunctiva and upon entering into the aqueous humor, iris, and ciliary body. Proteases or peptidases are primarily responsible for hydrolysis of amide linkage in

Palpebral conjunctiva

Lacrimal gland

Bulbar conjunctiva Aqueous humor

(anterior chamber)

peptides or peptide-based prodrugs. These enzymes are classified as either ‘endo-’ or ‘exo-’ depending on whether they cleave internal or external peptide bonds. The aminopeptidase activity is the highest in the corneal epithelium

and iris-ciliary body followed by conjunctiva and corneal stroma.

Kashi and colleagues have shown that aminopeptidases, dipeptidyl peptidase, and dipeptidyl carboxylpeptidase are

involved in hydrolysis of enkhaphilins in rabbit oculartissue homogenates including conjunctiva, corneal stroma, iris ciliary body, lens, and tears.

Prodrugs targeted toward membrane transporters expressed on the epithelial cells are perhaps the most exciting of all the current drug-delivery strategies. Epithelial cells express various nutrient transporters and receptors on their membrane surface. Analogs or prodrugs targeted toward these transporters can significantly enhance the absorption of poorly permeating therapeutic agents. Such prodrugs are recognized by membrane transporters as natural substrates and are translocated across the epithelial membranes. Once inside the cell, the conjugate will release the parent drug by enzymatic hydrolysis (Figure 2). Various nutrient transporters are expressed on the cornea and conjunctiva. Their utility in drug delivery will be discussed in subsequent sections.

Role of Cornea in Topical Drug Delivery

Cornea is the outermost avascular and transparent domeshaped structure of the eye. It consists of five layers (in the direction from anterior to posterior): epithelium, Bowman’s layer, stroma, Descemet’s membrane, and corneal endothelium (Figure 3). The lipoidal corneal epithelium is comprised of five to six layers of tightly adherent columnar cells with tight-junction proteins called zonulae occludens – acting as a major barrier to hydrophilic drugs. On the other hand, the stroma – which is comprised of 90% water – lies directly beneath the corneal epithelium and acts as a rate-limiting barrier to lipophilic drugs. Thus,

even if a molecule is sufficiently lipophilic to rapidly cross the epithelium, penetration through the stroma is still rate limiting. In addition, the physicochemical properties of the drug itself limit its permeability across the cornea. More recently, the expression of multidrug resistance proteins such as the P-glycoprotein (P-gp) and multidrug- resistance-associated proteins (MRPs) has been reported on rabbit and human corneal epithelium. They have gained attention lately, since majority of the drug molecules applied topically have been categorized as substrates to one or more of these efflux pumps. In fact, P-gp and MRP-2 localized on the rabbit corneal epithelium can act as a barrier to in vivo drug absorption through cornea.

Lipophilic prodrug derivatization has been considered as a viable strategy to enhance transcorneal permeation of ocular therapeutic agents. High octanol/water coefficient of these prodrugs can facilitate permeation across the corneal epithelium. The water-laden stroma, in turn, does not act as a barrier to the regenerated hydrophilic parent drug – thereby enhancing the overall permeability of the prodrug across cornea. Esterase activity has been reported to be the highest in iris-ciliary body followed by cornea and the aqueous humor. Even though high levels of esterases are reported in the iris-ciliary body, the activity in the cornea is highly relevant since cornea acts as a major permeation pathway to these lipophilic ester prodrugs. Bulk of esterase-mediated hydrolysis takes place in the corneal epithelium where the esterase activity is about 2 times to that of stroma and endothelium.

Lipophilic ester prodrug design has been employed for a variety of ocular therapeutic agents, which suffer from poor ocular absorption. Esterification of prostaglandin F2a