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

(PGF2a) analogs which exhibit higher potency than PGF2a have resulted in blockbuster drugs such as bimatoprost, travoprost, latanoprost, and isopropyl unoprostone. Travoprost, latanoprost, and isopropyl unoprostone are isopropyl esters of PGF2a analogs, whereas bimatoprost is an ethyl amide prodrug (Figure 4). The free acid form of all the above prodrugs has shown poor permeability across cornea, thus obviating the need for lipophilic ester prodrug design. Such strategy is particularly applicable to b-blockers such as timolol – widely used in the treatment of glaucoma – which suffers from high incidence of cardiovascular and respiratory side effects due to systemic absorption of the topically administered dose. Lipophilic prodrug derivatization of timolol to acetyl, propionyl, and butyryl ester prodrugs resulted in corneal permeabilities 2–3 times higher than timolol. Moreover, the enhanced corneal permeation leads to a fourto sixfold increase in the aqueous humor concentrations. Better corneal permeation and high aqueous humor concentration of lipophilic ester prodrugs of timolol consequently resulted in a twofold reduction in the topical dose. Reduced dose can, in turn, decrease the concentration in the systemic circulation, thereby reducing the incidence of cardiovascular and respiratory side effects.

The lipophilic ester prodrug design has been extended to antiviral agents such as acyclovir (ACV) and ganciclovir (GCV). These are highly potent against Herpes simplex virus (HSV). Currently available therapy for HSV keratitis involves the use of a 1%-trifluorothymidine (TFT) solution. However, long-term treatment with TFT raises potential concerns due to its high cytotoxicity. ACV is a potent candidate effective against HSV which is available as a 3% ophthalmic ointment. However, due to various problems associated with the use of ointments in the eye, it has not been approved in the United States. Ocular bioavailability of these compounds is extremely limited because of poor corneal permeability. Corneal permeability coefficients increase with increasing lipophilicity for monoester prodrugs of GCV, with the optimal prodrug form having three or four carbon atoms in the side chain. The apparent permeability (Papp) of the valerate-ester prodrug of GCV was sixfold higher than the parent drug GCV, across the cornea. Acylation of ACV also led to improved corneal permeation of the parent drug. There is a linear relationship between the corneal permeability coefficient and the octanol/water partition coefficient with a positive slope, except for ACV isobutyrate – which displayed an anomalously low corneal permeability despite improved lipophilicity. The anomaly with branched alkyl side chain could be explained due to enhanced stability of these prodrugs toward esterases present in the corneal epithelium. The branched side chain offers steric hindrance for enzyme accessibility. The concept has been proven where the stability of homologous series of oxprenolol esters increases with

increasing carbon chain length of the side chain. It has been shown that hydrolysis rates are moderate for O-propionyl, O-butyryl, and O-valeryl prodrugs of oxprenolol, with O-acetyl-oxprenolol being highly unstable and O-pivaloyl-oxprenolol being highly stable (Table 1). Again, the steric hindrance offered by the bulky tertiary butyl group in the pivaloyl derivative has been cited as the primary reason for superior enzymatic stability. However, all of these prodrugs described above are highly lipophilic and possesses very low aqueous solubility to be formulated into aqueous drops for topical administration. Reports indicate that the aqueous solubility of the lipophilic ester prodrugs decrease with increasing carbon side-chain length. Low aqueous solubility has been considered as a major drawback for formulating these lipophilic ester prodrugs into eyedrops. Thus, for a compound to be effective topically and to be formulated into eyedrops, it must possess sufficient hydrophilicity and at the same time exhibit sufficient permeability across the cornea to reach therapeutic levels.

Recently, transporter-targeted prodrug approach has received significant attention and a number of membrane transporters have been discovered in various ocular tissues such as the cornea, conjunctiva, and retina. These transporters are involved in the translocation of essential nutrients and xenobiotics across biological membranes. Ocular transporters include carriers for peptides, amino acids, glucose, lactate, and nucleosides/nucleobases and are primarily localized on the corneal epithelium, corneal endothelium, retinal pigmented epithelium (RPE), and retinal capillary endothelium. Prodrugs or analogs designed to target these transporters can significantly enhance the absorption of poorly permeating parent drug. Both solubility and the desired membrane permeability can be achieved by proper selection of the promoiety. Such prodrugs are recognized by the membrane transporters as substrates and are translocated across the epithelia. Subsequently, the prodrugs are enzymatically cleaved to release the parent drug and free ligand which in most cases is a nutrient and nontoxic.

Of late, such transporter-targeted prodrug design has been applied to the antiviral drugs ACV and GCV. The existence of an oligopeptide transport system (PEPT1) on the rabbit corneal epithelium has been demonstrated which can transport peptidomimetic prodrugs of acyclovir. Permeation of valine-ester prodrug of acyclovir (L-Val-ACV) across the cornea has been found to be much higher than that of the parent drug ACV (Table 2). The transport of L-Val-ACV is saturable at higher concentrations, pH dependent, and competitively inhibited by other known PEPT1 substrates indicating its translocation by PEPT1 present on the cornea. This result prompted the investigators to synthesize a series of water-soluble dipeptide prodrugs of acyclovir targeting the peptide transport system on the cornea to improve the ocular bioavailability. The structures of ACV, valine-based amino acid ester of

Table 2 Permeability of dipeptide prodrugs of ACV across freshly excised rabbit cornea

aControl

All values are expressed as mean SD. Statistically significant difference between control and test is represented by * (p < 0.05). ACV, acyclovir; VACV, val-acyclovir; VVACV, val-val-acyclovir; GVACV, gly-val-acyclovir; YVACV, tyr-val-acyclovir; VYACV, val-tyr-acyclovir.

Data from Anand, B. S., Nashed, Y. E., and Mitra, A. K. (2003). Novel dipeptide prodrugs of acyclovir for ocular herpes infections: Bioreversion, antiviral activity and transport across rabbit cornea. Current Eye Research 26: 151-163, with permission from Taylor & Francis.

ACV (VACV), and valineand glycine-based dipeptide conjugate of acyclovir – valine–valine–acyclovir (VVACV) and glycine–valine–acyclovir (GVACV) are shown in Figure 5. VACV is found to be hydrolyzed primarily by esterases to release ACV, whereas VVACV is hydrolyzed initially by aminopeptidases to release VACV, which is further acted upon by esterases to release ACV (Figure 6). Direct conversion of VVACV to ACV appears to be minimal. These prodrugs exhibited excellent solution stability and solubility allowing formulation into suitable eyedrops. All dipeptide prodrugs of ACV exhibited enhanced transcorneal permeability resulting in higher ocular bioavailability in rabbits, with GVACV being the highly permeable prodrug (Table 2). The prodrugs also exhibit higher antiviral efficacy against HSV epithelial keratitis and stromal keratitis and are less cytotoxic and more effective than trifluorothymidine (TFT) – a current drug of choice.

In addition to peptide transporters, even amino acid transporters such as ASCT1 (Naþ-dependent neutral amino acid transporter) and B0,þ (Naþ-dependent neutral and cationic amino acid transporter) have been explored for ocular delivery of ACV. A series of amino acid ester prodrugs including alanine-ACV, serine-ACV, isoleucine-ACV,

g-glutamate-ACV, and valine-ACV were synthesized and evaluated by Katragadda and colleagues for in vivo corneal absorption against the parent drug, ACV. Results showed that the amino acid-ester prodrug, serine-ACV – owing to its enhanced stability – exhibited higher area under the curve, Cmax and Clast values, in comparison to ACV and seemed to be a promising candidate for the treatment of ocular HSV infections. Gunda and colleagues have applied similar chemical derivatization to GCV – an acyclic guanosine analog. GCV has shown to exhibit excellent antiviral activity against the herpes viruses but suffers from poor corneal permeability due to its hydrophilicity. Hence, in order to enhance the corneal permeability and ocular bio availability, dipeptide monoester prodrugs of GCV targeting the peptide transporter on the corneal epithelium were synthesized. Among the synthesized prodrugs, tyrosine– valine–GCVand valine–valine–GCV, exhibited significantly higher transcorneal permeability ex vivo and in vivo leading to higher ocular bioavailability when compared to GCV.

Role of Conjunctiva in Ocular Drug Delivery

Conjunctiva is a transparent, highly vascularized mucous membrane that covers the sclera and lines the inner surfaces of eyelids. It covers almost 80% of exposed ocular surface and is comprised of many small blood vessels and tiny secretory glands. These glands produce tear film that lubricates and protects the eye during its movement in the socket. Three different types of conjunctival membranes have been identified based on its location. Palpebral or tarsal conjunctiva is the one lining the eyelids. Bulbar or ocular conjunctiva is a semipermeable and colorless membrane covering the eyeball, over the sclera. Fornix conjunctiva is where the inner part of the eyelids and the eyeball meet. It is loose and flexible, allowing free movement of the lids and eyeball. The tissue consists of pseudostratified columnar epithelium rich in goblet cells and it contains ductules of main lachrymal gland, accessory lachrymal glands, and lymphoid follicles. The space between the palpebral and bulbar conjunctiva is called the conjunctival cul-de-sac. Conjunctiva is typically composed of two layers, an outer epithelium and underlying stroma. The epithelium consists of 5–15 layers of

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stratified epithelial cells and is covered with microvilli. The stroma loosely attaches to the underlying sclera. It contains all the lymphatics and blood vessels.

The larger surface area and pore density coupled with expression of various nutrient transporters listed in the subsequent sections makes the conjunctiva more amenable for topical drug delivery. The surface area of the conjunctiva is almost 9 and 17 times larger than that of cornea in rabbits and humans, respectively. Compared to cornea, the paracellular pore size as well as the pore density in conjunctiva is larger. Due to such increased pore size and density small peptides and oligonucleotides can permeate across conjunctival pores. However, for

hydrophilic drugs instilled topically, the lipophilic conjunctival epithelium acts as rate-limiting barrier for drug absorption. In addition, an external enzymatic barrier in the conjunctival epithelium – specifically the proteases – restricts the penetration of peptide drugs like enkephalins, substance P, and insulin. One of the major disadvantages associated with conjunctival absorption following topical administration is drainage of drug molecules by conjunctival blood vessels into the systemic circulation. Moreover, it has been shown that the conjunctiva expresses efflux transporters including P-gp on the apical side of conjunctival epithelium. These can limit the absorption of several fluoroquinolones such as levofloxacin, gatifloxacin, and

Aminopeptidases

ACV

Figure 6 Metabolic pathway of valine–valine–acyclovir (VVACV) to valine-based amino acid ester of acyclovir (VACV) and acyclovir (ACV). From Anand, B. S., Nashed, Y. E., and Mitra, A. K. (2003). Novel dipeptide prodrugs of acyclovir for ocular herpes infections: Bioreversion, antiviral activity and transport across rabbit cornea. Current Eye Research 26: 151–163, with permission from Taylor & Francis.

grepafloxacin instilled topically. Thus, improving the drug absorption across conjunctiva is one of the major challenges needed to be overcome. Based on the target tissue, conjunctival drug delivery can be divided into subconjunctival and transconjunctival delivery.

Transconjunctival Pathway

In this route, agents can be targeted toward the conjunctiva for treatment of local conjunctival infection as well as other anterior chamber diseases such as dry eye syndrome and glaucoma. Transconjunctival absorption could also result in higher concentrations in posterior ocular tissues through conjunctival–scleral pathway. However, subconjunctival pathway is widely exploited to deliver drugs to posterior ocular segment.

Hydrophilic molecules can permeate through paracellular pathway between epithelial cells through the tight junctions. However, their penetration will be extremely low due to the small surface area of paracellular pathway compared to transcellular route. Transconjunctival drug penetration can be enhanced by increasing the lipophilicity of the drug molecule through chemical modification, mostly as prodrugs or analogs. Propranolol – with a log partition coefficient between octanol and water (log P) of 3.21 – is absorbed through cornea and conjunctiva up to tenfold greater than a hydrophilic drug of similar size, for example, sotalol with a log P of 0.62.

Prodrug strategy has not been exploited in conjunctiva as extensively as in cornea. As discussed earlier, in vivo activation of the prodrug is very essential for the success of this approach. As ester and amide linkages are widely utilized in prodrug design, it is important to know the activities of esterases and amidases in conjunctiva. As mentioned before, esterase and aminopeptidase activity has been reported in conjunctiva along with the cornea and iris-ciliary body. These play a role in hydrolysis of ester and amide bases prodrugs in the conjunctiva.

The peptidases can also hydrolyze short-chain, biologically active peptides such as methionine and leucine enkephalins. There is good number of studies demonstrating esterases/amidases targeted prodrug approach to enhance drug absorption across conjunctiva. A few of them are discussed below.

PGs can lower intraocular pressure (IOP) in openangle glaucoma. However, due to the low permeability of PGF2a across the cornea, a relatively high concentration of PGF2a is necessary for effective IOP reduction. This can result in conjunctival hyperemia, ocular discomfort, headaches, and other side effects. Chen and colleagues have evaluated lipophilic PGF2a ester prodrugs for improving the permeability of PGF2a following topical administration. A series of lipophilic esters such as PGF2a 1-isopropyl, 1, 11-lactone, 15-acetyl, 15-pivaloyl, 15-valeryl, and 11, 15-dipivaloyl esters have been evaluated for transport and bioreversion in rabbit cornea, conjunctiva, and iris-ciliary body (Figure 7). All of the prodrugs penetrated the rabbit cornea and conjunctiva faster than PGF2a except the 15-acetyl ester prodrug, which is equally permeable as PGF2a across conjunctiva. However, a direct relationship is not observed between the degree of apparent permeability and prodrug lipophilicity. The two most lipophilic prodrugs – the 15-valeryl and 11, 15-dipivaloyl esters – were less permeable in the cornea and conjunctiva than other prodrugs with comparatively lower lipophilicity. The high permeabilities of 1-isopropyl ester and 1, 11-lactone is attributed to their state of ionization at the site of absorption where they exist more in unionized form. The prodrug 1, 11-lactone hydrolyzed at a slower rate in the ocular tissues probably due to the intracyclic ester linkage. The bulky pivaloyl group in 15-pivaloyl prodrug rendered it enzymatically more stable than the 1-isopropyl and other 15-monoester prodrugs. Bulky moiety (steric hindrance) might have impeded access of esterases to hydrolyze the pivaloyl ester linkage. Similarly, the prodrug with two pivaloyl groups – 11, 15-dipivaloyl prodrug – is much more stable as expected. Thus, the size and structural branching of the promoiety can influence enzymatic hydrolysis of prodrugs which, in turn, can further determine the permeability across tissues.

Corticosteroids find applications in the treatment of uveitis and postsurgical inflammation. However, these drugs can cause significant adverse effects such as increase in IOP, cataract, and herpetic reactivation. Dexamethasone (DX) is the one drug mostly indicated for ocular corticosteroid treatment. Several lipophilic esters of dexamethasone prodrugs have been evaluated for their ocular permeability and bioreversion in bovine conjunctival epithelial cell line (BCEC) and isolated rabbit cornea (Figure 8). This study is mainly aimed at selecting an optimum prodrug that improves the delivery of dexamethasone to the target tissue with minimum permeation across other ocular tissues to reduce adverse effects. The permeability

of these dexamethasone prodrugs correlated well with their lipophilicity until a maximum value is reached, which corresponded to dexamethasone butyrate (log P ¼ 3.95). DSP and DSB prodrugs are hydrophilic and thus do not permeate across BCEC as expected. Other prodrugs exhibited higher permeability with increase in lipophilicity till DBU, after which it started to plateau as observed with DVA (Figure 9). A similar trend has been observed across the cornea with exceptions at the extreme ends of log P values (DSP and DVA) which has been attributed to the rate of hydrolysis of prodrugs in corneal tissue and BCEC an epithelial layer. DVA being highly lipophilic gets absorbed into corneal

epithelium and its further permeation is limited by hydrophilic corneal stroma unless it is hydrolyzed to relatively more hydrophilic dexamethasone. However, the hydrolysis of DVA to dexamethasone is very poor in cornea, confining the prodrug in the corneal epithelium (Table 3). This may be optimum for delivery of dexamethasone to cornea, where the prodrug in the corneal epithelium can hydrolyze slowly and sustain the release. Thus, it reduces the drug concentrations in aqueous humor and other intraocular tissues minimizing its side effects. Unlike in BCEC, DSP has been found to hydrolyze rapidly in the cornea creating a concentration gradient for higher absorption into cornea. DBU is the

prodrug that is completely hydrolyzed both in BCEC and cornea and also has tremendously improved the permeability of dexamethasone. This may be more suitable prodrug to deliver dexamethasone to other intraocular tissues. These studies clearly show that both the promoiety as well as the ability of the prodrug to get hydrolyzed are important in designing an optimum prodrug in ocular drug delivery.

A series of alkyl, cycloalkyl, and aryl ester prodrugs of timolol has been evaluated to examine the effect of enzymatic lability of prodrugs on corneal and conjunctival penetration of timolol. Straight-chain alkyl and the unsubstituted cycloalkyl esters hydrolyzed more rapidly than their corresponding branched-chain and substituted analogs as well as the aryl esters. This might be due

Figure 9 Permeability rates of dexamethasone esters through BCEC (filled squares) and excised cornea (empty circles) versus log P. Left to right, 21-sodium phosphate ester (DSP), 21-metasulfobenzoate ester (DSB), dexamethasone (DX), 17-propionate ester (DPR17), 21-acetate ester ( DAC), 21-propionate ester (DPR), 21-butyrae ester (DBU), and 21-valerate (DVA). From Civiale, C., Bucaria F., Piazza S., et al. (2004). Ocular permeability screening of dexamethasone esters through combined cellular and tissue systems. Journal of Ocular Pharmacology and Therapeutics 20: 75–84, with permission from JOPT.

to free accessibility of the ester linkage to esterases in straight-chain alkyl esters. The slower hydrolysis of branched chain alkyl esters might be due to steric hindrance to hydrolyzing enzymes. As expected, the esters with enzymatically labile straight-chain alkyl chains penetrated the cornea and conjunctiva at faster rates than the esters with branched alkyl chains that are less labile toward enzymatic hydrolysis. Thus, this study has shown that the rate of enzymatic hydrolysis can highly influence the corneal/ conjunctival absorption of prodrugs.

Carrier-mediated transport represents an area of growing interest to pharmaceutical scientists. These transport systems play an important role in absorbing nutrients as well as their mimetic drugs. Transporter-targeted prodrug delivery can be an effective strategy to improve the permeability of poorly absorbed drugs, as conjunctiva has been shown to express various transporters for nutrients such as amino acid, peptides, L-lactate, and nucleosides. These transporters can be utilized for improved drug absorption across the conjunctiva. However, such a strategy has not been explored to a large extent.

Functionally active sodium-dependent, carriermediated, monocarboxylate transport system has been reported on the mucosal or tear-side of the rabbit conjunctival epithelium. It has been shown to recognize and translocate nonsteroidal anti-inflammatory drugs (NSAIDs) and fluoroquinolone antibiotics administered topically. The conjunctiva expresses Naþ-glucose transporter (SGLT1) on the mucosal side and a sodium-dependent nucleoside transporter has been characterized that can be targeted by nucleoside mimetic antivirals on conjunctiva. Various amino acid-transporter systems including L-lysine and B0,þ are present on the apical side of rabbit conjunctiva. Among various transporters exploited for drug delivery, peptide transporters (PEPT1 and 2) have become popular due to their high capacity and wide substrate specificity. These play an important role in the translocation of diand tripeptides and peptidomimetic drugs

across various tissues. A proton-coupled dipeptide transporter process has been reported to be present on pigmented rabbit conjunctiva. A proton-coupled, saturable, and temperature-dependent uptake of a dipeptide on rabbit conjunctival epithelial cells (RCEC) was reported. However, the expression level of the dipeptide transporters seems to be rather low in conjunctiva. Overall, the presence of a variety of influx transporters on the conjunctiva offers an immense potential for targeted delivery after topical prodrug administration.

Subconjunctival Delivery

This approach of drug delivery is more popular and safer and less invasive than intravitreal injection. Moreover, it offers the potential advantage of localized, sustained delivery in the treatment of ocular diseases affecting the posterior segment such as age-related macular degeneration (AMD) and diabetic retinopathy. As this route can circumvent both the corneal and conjunctival barriers, it has immense potential for delivery of both small molecules and macromolecular drugs to posterior ocular tissues including choroid and retina. Moreover, sclera is much more permeable than conjunctiva. Drug delivery by this route exploits the large surface area, easy accessibility, and relatively high permeability of sclera. By this route, sustained intraocular therapeutic drug concentrations can be achieved without surgical implantation of controlled-release drug-delivery implant in the vitreous humor or by repeated intravitreal/periocular injections. In this regard, a subconjunctival injection of drug-loaded nanoparticulate system offers potential alternative. These systems can release the drug in a sustained manner as well as achieve higher drug concentrations in the target tissues. Drug-loaded nanoparticulate systems prepared with biodegradable polymers have been widely used in sustaining the drug release. Nanoparticles are usually taken up into the cell by endocytosis which will significantly enhance the uptake of nanoparticles into the targeted cells. Moreover, receptor-mediated endocytosis have attracted attention due to high capacity and targeting feasibility. Expression of various receptors by RPE will enable this strategy to be successfully applied for subconjunctival administration of receptor-targeted nanoparticles. In this strategy, a promoiety is conjugated to the polymer, which is used to prepare these nanoparticles. It is also ensured that these targeting promoieties are present on the surface of nanoparticles, such that it is recognized by a specific receptor present on the RPE and undergo receptor-mediated endocytosis. Thus, drug loaded nanoparticles improve drug absorption into RPE, enable targeting, and sustain the release for prolonged periods of time.

In addition to this, a sustained drug release can also be achieved by dispersing the drug/prodrug in a thermosensitive gelling polymer. These are the polymers that are

liquids at room temperature and gels at body temperature. Drug/prodrug can be mixed homogenously in the polymer solution and injected subconjunctivally. Upon injection, the polymer solution gels and sustains the release of drug. The drug loading and release can be optimized to maintain therapeutic concentrations for prolonged periods of time.

Conclusion

Prodrugs have proven to be an effective strategy for drug delivery to the anterior segment. Several examples of successfully marketed ophthalmic prodrugs including dipivalyl ester of epinephrine (dipivephrine) are available. In addition, ester prodrugs of PGF2a analogs such as bimatoprost, travoprost, and latanoprost are highly effective. However, the application of this strategy to a particular drug molecule depends upon its chemical structure. The drug molecule needs to have a specific functional group such as carboxyl/hydroxyl/amine group to facilitate the conversion to an ester or amide-based prodrug. Despite this, a significant amount of research needs to be performed to examine the feasibility of extending the prodrug strategy to deliver macromolecular drugs like proteins and antibodies which have been highly effective in treatment of various ocular diseases, especially the AMD.

See also: Cornea Overview; Corneal Angiogenesis; Corneal Endothelium: Overview; Corneal Epithelium: Transport and Permeability; The Corneal Stroma; Overview of Electrolyte and Fluid Transport Across the Conjunctiva.

Further Reading

Anand, B. S., Nashed, Y. E., and Mitra, A. K. (2003). Novel dipeptide prodrugs of acyclovir for ocular herpes infections: Bioreversion, antiviral activity and transport across rabbit cornea. Current Eye Research 26: 151–163.

Chang, S. C., Bundgaard, H., Buur, A., and Lee, V. H. (1987). Improved corneal penetration of timolol by prodrugs as a means to reduce systemic drug load. Investigative Ophthalmology and Visual Science

28: 487–491.

Chien, D. S., Sasaki, H., Bundgaard, H., Buur, A., and Lee, V. H. (1991). Role of enzymatic lability in the corneal and conjunctival penetration of timolol ester prodrugs in the pigmented rabbit. Pharmaceutical Research 8: 728–733.

Chien, D. S., Tang-Lio, D. D., and Woodward, D. F (1997). Ocular penetration and bioconversion of prostaglandin F2alpha prodrugs in rabbit cornea and conjunctiva. Journal of Pharmaceutical Sciences

86: 1108–1186.

Civiale, C., Bucaria, F., Piazza, S., et al. (2004). Ocular permeability screening of dexamethasone esters through combined cellular and tissue systems. Journal of Ocular Pharmacology and Therapeutics

20: 75–84.

Geraldine, C. and Jordan, M. (1998). How an increase in the carbon chain length of the ester moiety affects the stability of a homologous series of oxprenolol esters in the presence of biological enzymes.

Journal of Pharmaceutical Sciences 87: 880–885.

Gunda, S., Hariharan, S., and Mitra, A. K. (2006). Corneal absorption and anterior chamber pharmacokinetics of dipeptide monoester prodrugs of ganciclovir (GCV): In vivo comparative evaluation of these prodrugs with Val-GCV and GCV in rabbits. Journal of Ocular Pharmacology and Therapeutics 22: 465–476.

Horibe, Y., Hosoya, K., Kim, K. J., and Lee, V. H. (1998). Carriermediated transport of monocarboxylate drugs in the pigmented rabbit conjunctiva. Investigative Ophthalmology and Visual Science

39: 1436–1443.

Hosoya, K., Lee, V. H., and Kim, K. J. (2005). Roles of the conjunctiva in ocular drug delivery: A review of conjunctival transport mechanisms and their regulation. European Journal of Pharmaceutics and Biopharmaceutics 60: 227–240.

Hughes, P. M. and Mitra, A. K. (1993). Effect of acylation on the ocular disposition of acyclovir. II: Corneal permeability and anti-HSV 1 activity of 20-esters in rabbit epithelial keratitis. Journal of Ocular Pharmacology 9: 299–309.

Kashi, S. D. and Lee, V. H. (1986). Hydrolysis of enkaphilins in homogenates of anterior segment tissues of the albino

rabbit eye. Investigative Ophthalmology and Visual Science 27: 1300–1303.

Katragadda, S., Gunda, S., Hariharan, S., and Mitra, A. K. (2008). Ocular pharmacokinetics of acyclovir amino acid ester prodrugs in the anterior chamber: Evaluation of their utility in treating ocular

HSV infections. International Journal of Pharmaceutics 359: 15–24. Lee, V. H. (1983). Esterase activities in adult rabbit eyes. Journal of

Pharmaceutical Sciences 72: 239–244.

Stratford, R. E., Jr. and Lee, V. H. (1985). Ocular aminopeptidase activity and distribution in the albino rabbit. Current Eye Research 4: 995–999.

Tirucherai, G. S., Dias, C., and Mitra, A. K. (2002). Corneal permeation of ganciclovir: Mechanism of ganciclovir permeation enhancement by acyl ester prodrug design. Journal of Ocular Pharmacology and Therapeutics 18: 535–548.

Glossary

Cre – The phage recombinase that catalyzes cyclization recombination of LoxP elements.

EGF – The epidermal growth factor which binds to its receptor, EGFR.

FGF – The family of growth factors involved in angiogenesis, wound healing, and development. Keratins – The family of structural proteins that are tough and insoluble. Keratin 12 is specific to the cornea.

Keratocan – A member of keratan sulfate proteoglycans that is important for transparency of the cornea.

LoxP – The locus of crossover within P1 phage.

Reverse tetracycline transcriptional activator (rtTA) – Binds to Tet operator sequence 7 and activates transcription of the target gene in the presence of tetracycline (Tet-on system).

Tetracycline operator element – The tetracyclineregulated promoter that contains tetracycline operator sequences, which regulate expression of a downstream gene.

Introduction

Transgenesis, that is, the insertion of an exogenesis gene into an organism such that the gene is transmitted to the offspring, and gene targeting are among the most important biological techniques developed in the twentieth century. The studies of genetically modified mutant mice by transgenesis and gene targeting are of great value to elucidate the pathophysiology of altered gene functions and have greatly increased our knowledge of normal physiology and diseases in humans. They not only provide the means for the generation of animal models that are used to examine the pathogenesis of human diseases caused by altered genetic functions, but also allow the development of gene and cell therapy strategies to treat diseases. For example, the application of transgenesis and gene-targeting techniques opens the door of targeted introduction of genes to cells of diseased tissues, that is, gene therapy. Thus, lost cellular and tissue functions can be restored and diseases are cured.

Transgenesis

Transgenesis through microinjection of cloned DNA into fertilized mouse eggs was first accomplished in the laboratories of Brinster, Costantini, Ruddle, Mintz, and Wagner. The availability of a cell-type-specific promoter is a prerequisite for the success of creating transgenic mouse lines that exhibit altered phenotypes caused by the presence of such reporter gene product in tissues of interest. Since then, the technology of transgenesis has advanced to create inducible transgenic mouse lines in which a reporter gene has a unique spatial and temporal expression pattern by administering antibiotics, hormones, and pheromones to experimental animals. The system usually consists of two transgenic mouse lines, one of which employs a tissue-specific promoter for the expression of a transgene that encodes a fusion protein of a transcription factor and hormone receptor and antibiotic suppressor. The other is a transgenic mouse carrying a reporter gene following the responsive elements of a transcription factors and suppressor. In the bitransgenic offspring from the mating of the two transgenic mice, the reporter genes can be turned on and off when ligands bind to the fusion proteins of transcription factor/receptor and transcription factor/suppressor fusion proteins. For example, tet-ON and tet-OFF system in overexpression of tet-O- FGF7 by epidermal epithelium under the control of reverse tetracycline transcription activator (rtTA) driven by keratin 5 (K5) and K14 promoters of bitransgenic K5-rtTA/tet-O- FGF7 and/or K14-tTA/tet-O-FGF7 mice.

Gene Targeting

In late 1980, Doetschman et al. and Thomas et al. demonstrated that the mouse genome could be modified in vitro in embryonic stem cells by homologous recombination. It was subsequently demonstrated that the modified mouse genome could be transmitted to offspring through injection of such genetically modified embryonic stem cells into blastocysts. Since then, many gene-targeting strategies have been developed not only to ablate functional genes (i.e., knockout), but also to replace target genes with another functional gene and/or a mutant gene of interest (knock-in). These strategies allow us to examine gene function in experimental mouse lines that mimic pathogenesis of human diseases and yield useful information, leading to a better understanding of normal physiology and pathophysiology in humans.

However, many of the altered genes often lead to embryonic lethality and detrimental effects on animal