The Conjunctival Barrier in Ocular Drug Delivery

13The Conjunctival Barrier in Ocular

Drug DeliveryHovhannes J. Gukasyan, Kwang-Jin Kim, and Vincent H.L. Lee

Abstract Within the context of topical and local drug delivery to the eye,the mammalian conjunctiva functions as a unique biological barrier. Variousmodel systems as in vitro tools have been refined and validated over the years toassess drug absorption across the conjunctiva. Passive and active drug transportas well as endocytic routes of transconjunctival drug permeation have beenextensively characterized. The subconjunctival space offers the possibility ofdelivering multiple types of sustained release formulations to the eye. Addition-ally, the concept of transscleral/conjunctival iontophoresis has been proven asa viable option for topical, relatively noninvasive delivery of select categoriesof molecules. Furthermore, a number of ocular surface disorders have beenassociated with conjunctival phenotypes, making this tissue a potential drugtarget. The aim of this chapter is to facilitate the understanding of conjunctival(transport) physiology and establishment of in vitro models of conjunctivavalidated for predicting drug delivery to the eye in health and disease. Themodels are evaluated in terms of assessing their electrical, morphological, andpermeability properties as well as expression profiles of endogenous activetransporter(s) and marker enzyme(s). Finally, we provide protocols and meth-ods, as an appendix to this chapter, to be used for in vitro studies of theconjunctiva for testing preclinical biopharmaceutics.

Keywords: Ocular drug delivery; Noncorneal route; Transporters; Ion-tophoresis; Endocytosis; Dry eye; Inflammation; Subconjunctival injections

Abbreviations

Ad5 Adenovirus type 5AIC Air-interfaced cultureATB Na+/Cl−-coupled broad specificity amino acid transporter B0,+ATP Adenosine triphosphatecAMP Cyclic adenosine monophosphateCFTR Cystic fibrosis transmembrane conductance regulatorGSH GlutathioneIsc Short-circuit current

307

308 H. J. Gukasyan et al.

L-NA NG-nitro-L-arginineNO Nitric oxideNOS Nitric oxide synthasePD Potential differenceP-gp P-glycoproteinRCEC Rabbit conjunctival epithelial cellsTEER Transepithelial electrical resistance

13.1. Introduction to the Ocular Surface and the RelativeContribution of the Conjunctiva

The conjunctiva is a thin, mucus-secreting, relatively well vascularized (com-pared to avascular cornea) tissue that covers ∼80% of the ocular surface. Itcomprises the inner surface of the eyelids and is part of the anterior scleraending where the cornea begins. While the conjunctiva has been long thoughtto play a simple protective role in the eye by functioning as a passive physicalbarrier, it is an intricate and heterogeneous tissue which participates in themaintenance of tear film stability due to the mucus secreted by its residentgoblet cells [1, 2]. Studies performed in recent years have characterized addi-tional functional and practical features of the conjunctiva, that is, contributingto the regulation of electrolyte and fluid balance in the microenvironment ofits mucosal surface as well as providing a unique conduit for drug deliveryto the anterior and posterior segments of the eye when drugs are appliedtopically to ocular surfaces [3, 4]. The dynamic nature of conjunctiva wasdemonstrated from the identification of several transport mechanisms for Na+absorption from the conjunctival mucosa: Na+-glucose, Na+-amino acid, andNa+-nucleoside cotransporters, in addition to the predominant and active Cl−secretion. Permeability of the conjunctiva to a wide variety of hydrophilicand lipophilic molecules and drugs was also characterized in the recent past,which is detailed in a recent review [5]. Furthermore, multiple reports sub-stantiate mechanisms of conjunctival ion transport, a key defining property ofthis tissue. Pharmacological fingerprints of conjunctival ion transport includesignificant reduction by the serosal application of bumetanide, an inhibitorof Na+/K+/2Cl− cotransporter, the only known serosal entry pathway forCl− into conjunctival cells to date [6–8]. Net conjunctival Cl− secretion ismodulated by cyclic adenosine monophosphate (cAMP), protein kinase C, andCa2+, suggesting the presence of at least three mucosal Cl− exit pathways[8]. Moreover, the existence and apical localization of CFTR (cystic fibrosistransmembrane conductance regulator) was confirmed by electrophysiological,molecular, biological, and immunocytochemical methods in the pigmentedrabbit conjunctival epithelium [9, 10]. By contrast, metabolic enzymes or bio-chemical elimination mechanisms in the conjunctiva remain largely unknown.In addition to acting as a highly specialized secretory epithelium within thecontext of biochemical and metabolic capabilities, the conjunctiva also displaysesterase activity [11], and is likely to express isoforms of drug efflux pumps(e.g., P-glycoprotein, P-gp) [12]. Lastly, the vectorial secretion of glutathione(GSH, an abundant and ubiquitous antioxidant tripeptide) from conjuncti-val epithelial cells has been functionally characterized in both the primary

Chapter 13 The Conjunctival Barrier in Ocular Drug Delivery 309

Fornicialconjunctiva

Bulbarconjunctiva

Palpebralconjunctiva

Sclera

Cornea

Lacrimal gland

Up

per

eye

lid

Lower eyelidAnteriorsegment

Posteriorsegment

Figure 13.1 A cross-sectionalrepresentation of the ocular surface,highlighting the conjunctival tissue as athick black contour. Three distinct areasof the conjunctiva are labeled alongwith adjacent ocular tissues.

culture model and excised tissues of conjunctivas obtained from the pigmentedrabbit [13].

The ocular epithelium, obtained from other mammalian epithelial tissuesthat serve as primary barriers from the outside environment, is atypical instructure and function. Comprising relatively and proportionally a small areaof the total body surface, the ocular surface includes two continuous but verydifferent tissues of the conjunctiva and cornea. While the conjunctiva coversmost of the ocular surface area, only a small fraction (called the bulbar region)covers the anterior sclera, which has an air interface exposed to the environmentduring “open-eye” intervals (Figure 13.1). It continues through the fornicialand palpebral regions as highly vascularized, thin/semitransparent, elastic,and heterogeneous tissue (Figure 13.1). The cornea is a hydrophobic barriercontinuous with the bulbar conjunctiva (Figure 13.1), has a smaller relativesurface area, shows a clear transparency to light, exhibits a characteristichigh resistance to passive diffusion of ions and molecules, and withstandsagainst the intraocular pressure. Both the corneal and conjunctival epithe-lial tissues are constantly subjected to light irradiation, atmospheric oxygen,environmental chemicals, and physical abrasion. Each of these factors posesstress of primarily oxidative nature that can ultimately contribute to ocu-lar surface damage and disease, if left unchecked. Natural protective com-ponents like water-soluble antioxidants (e.g., vitamin C, L-cyst(e)ine, GSH,uric acid, pyruvate, and tyrosine), lipid-soluble antioxidants (e.g., tocopherolsand retinols), and highly specialized enzymes (e.g., superoxide dismutase,catalase, and GSH peroxidase) have all been identified in human tear fluidcollected at normal and stimulated secretion rates and are thought to serveas a frontline defense for the ocular surface tear film and underlying tis-sues. While mechanisms or glandular sources of these antioxidants have notbeen identified definitively, the conjunctiva may be a possible source dueto its dynamic properties of biochemical, electrophysiological, and secretoryfunctions.


13.2. Trans- and Sub-Conjunctival Ocular Drug Delivery

Although the cornea is the primary route of drug entry into the eye follow-ing topical administration, applied drugs also reach intraocular tissues byconjunctiva–scleral pathway. About 1–5% of a topically applied drug doseoften reaches the anterior segment of the eye. Therefore, subsequent diffusionof absorbed drugs to the posterior segment of the eye will often be relativelyinsignificant. Eye drops are eliminated from the precorneal area within 90 sand absorbed systemically by way of the highly vascular conjunctival stromaand nasolachrymal ducts. Despite several anatomical hurdles for intraoculardrug delivery by the conjunctival pathway, a number of critical discoverieshave been reported which highlight the importance of this route. Nipradilol,a β-blocker, and iganidipine, a Ca2+ antagonist, are good examples that havebeen reported to reach the posterior segment of the eye in preclinical species,respectively [5].

Improving the conjunctival drug permeability is one of the major challengesin ocular drug delivery. Enhancing transcellular drug penetration by increasingdrug lipophilicity through the use of prodrugs or analogs or improving para-cellular penetration by using enhancers to open tight junctions was reportedwith some success. The transcellular route significantly contributes toward theabsorption of lipophilic drugs, but is less significant for hydrophilic species.Propranolol, with a log partition coefficient between octanol and water (log PC)of 3.21, is absorbed through cornea and conjunctiva up to tenfold greater thana hydrophilic drug of similar size, for example, sotalol with a log PC of −0.62.A sigmoidal relationship, reported in various other tissues and in vitro epithelialcell culture systems, describes penetration of compounds through the conjunc-tiva and cornea, in a manner that an increase in lipophilicity is seen with arise in permeation, followed by apparent saturation at high lipophilicity. Sincehydrophilic drugs penetrate primarily via the paracellular pathway betweenepithelial cells through the tight junctions, their penetration area is extremelysmall compared to the surface area offered by transcellular route for absorp-tion of lipophilic species. However, most lipophilic drugs are prone to effluxmechanisms afforded by pumps (e.g., P-gp) localized on the apical side ofconjunctiva [5].

Delivery of biologic pharmaceuticals via the conjunctival route has beenstudied as an alternative to injections. Because of their hydrophilic nature,peptide and protein drugs permeate across ocular epithelia predominantlyvia the paracellular route. In conjunctival tissues, penetration through tightjunctions comprising the paracellular pathway is the rate-limiting step fordiffusion of macromolecule drugs. When this pathway is modeled to be pop-ulated with equivalent pores having a cylindrical shape, analysis suggeststhat conjunctiva may allow the passive restricted diffusion via such pores ofhydrophilic substances <20,000 Da. The theoretical radius of such equivalentpores is predicted to be ∼5.5 nm [14]. In addition, an external enzymaticbarrier also restricts the penetration of peptide drugs across the conjunctiva,where enkephalins, substance P, and insulin have been shown to significantlydegrade during penetration across the tissue when instilled to the mucosalfluid of conjunctivas mounted in modified Ussing chambers [15–17]. Withinthis context, the coapplication of protease inhibitors (e.g., camostat mesylateand leupeptin) in the mucosal fluid afforded the absorption of an increased


fraction of intact vasopressin across the excised pigmented rabbit conjunctiva[18]. Recent reports indicate that the conjunctival tissue may also be endowedwith endocytic vesicular routes for absorption of macromolecules [19, 20]. Itis important to note that the roles and their respective contributions of cave-olae, clathrin-coated pits, and other subcellular vesicle-associated elements inabsorption/secretion of protein drugs remain ambiguous at best [5].

Most of the prominent carrier-mediated transport systems, which have beenextensively characterized in the gastrointestinal tract, have been reported forocular absorption of amino acids, small peptides, sugars, monocarboxylates,organic cations, phosphates, bile acids, and several water-soluble vitamins[5]. These carrier-mediated transport systems can contribute toward the ocularabsorption of mimetic drugs. Since paracellular transport of drugs in ocularepithelia, especially the cornea and conjunctiva, is relatively limited in itscapacity, carrier-mediated drug transport systems endowed in the conjuncti-val epithelium (which has a larger surface area than the cornea) offer greatadvantages. Na+-coupled cotransport of D-glucose, L-arginine, nucleosides,and monocarboxylates as well as proton-dependent dipeptide cotransport haveall been reported in the conjunctival mucosa [5]. Utilizing these transportersfor absorption of (pro)-drugs has been proven conceptually feasible in local,ocular drug delivery. Nitric oxide synthase (NOS) inhibitors (e.g., NG-nitro-L-arginine methyl ester, NG-monomethyl-L-arginine, and NG-nitro-L-arginine,L-NA) are absorbed by the same active mechanism of the B0,+(ATB0,+) whichutilizes the essential amino acid L-arginine in the excised pigmented rabbitconjunctiva [21, 22]. As nitric oxide (NO) plays a cytostatic or cytotoxicrole in the conjunctiva and anterior uveal tract, transconjunctival delivery ofNOS inhibitors is a practical strategy to alleviate symptoms associated withNO overproduction. Since ATB0,+ is abundantly expressed in the mucosalaspect of conjunctiva and recognizes almost all of naturally occurring neu-tral and cationic amino acids as substrates, it may be a major drug deliveryfacilitator to target by rational design approaches. Therefore, there are a widerange of options for chemical modification of drugs through the applicationof prodrug approaches. For example, herpes simplex virus inhibitor, valacy-clovir, is transported as a substrate through mouse and human ATB0,+, invarious tissues including the eye. By virtue of analogous or similar aminoacid structures, ATB0,+ also affords the transport of acyclovir glutamic acidγ -ester [23]. Dipeptide transporters known to play a key role in the oralabsorption of certain β-lactam antibiotics and angiotensin-converting enzymeinhibitors are also present in the mucosal aspect of the conjunctiva (albeitwith relatively lower expression levels) [24–26]. Nucleoside transporters arereported to be expressed in the conjunctival mucosa [27], offering the potentialfor the targeting of nucleoside mimetic antiviral drugs. However, this classof transporter proteins exhibit narrow specificity for substrates, which mayentail a comparatively narrow option for chemical design in targeted drugdelivery [5].

The use of polymeric nanoparticles in the eye has gained considerable inter-ests in recent years. Ocular disposition, safety, efficacy, and pharmacokineticprofiles of various nanoparticles offer a wide range of application for thedelivery of many drugs used to treat common ocular disorders. Polymericnanoparticles have been utilized to enhance the performance of ibuprofen andcyclosporine, while reducing systemic side effect of carteolol compared with


commercial aqueous eye drops [28]. Poly-D,L-lactide-co-glycolide nanoparti-cles have been shown to be internalized (albeit direct proof of endocytosis islacking) in primary cultures of pigmented rabbit conjunctival epithelial cells(RCEC) [19]. As alluded above, although endocytic mechanisms of nanopar-ticles in the conjunctiva have not been fully characterized, this drug deliverystrategy may be a useful tool to apply in ocular delivery of novel therapeuticsthrough this tissue. The use of polymeric nano- and microparticles in subcon-junctival route of ocular drug delivery offers a unique method for enhancinglocal exposure level of drugs by maximizing and maintaining intraocular con-centrations with minimal frequency of dosing. Compared to direct intravitrealinjection, this approach is less invasive, although it requires the rational formu-lation design/small-scale engineering of both dimension and surface propertiesof the drug. Subconjunctival injection of polylactide microparticles (3.6 µm)led to a much higher budesonide concentration in retina and vitreous humorover 14 days, compared to solutions of dosing nanoparticles of smaller (345nm) size [29]. Collagen matrix and fibrin-based sealants provided a bettercontrolled release of cisplatin and carboplatin, compared to a simple drug solu-tions, attaining higher drug concentrations after subconjunctival administrationof rabbits in several intraocular tissues [30]. Since the sclera, an immediateunderlying tissue, is significantly more permeable than the conjunctiva, physi-cal and biological barriers of both cornea and conjunctiva are circumvented insubconjunctival injections [5].

Iontophoresis is a relatively noninvasive technique that uses electric currentsto drive introduction of ionized drug substances into tissues or across the tissuebarriers. Transcorneal or transscleral/conjunctival iontophoresis has been uti-lized in ocular drug delivery. Transcorneal iontophoresis effectively enhancesaqueous humor concentrations of drugs including gentamicin, tobramycin, andvidarabine monophosphate, but falls short of delivering these therapeutics inhigh enough concentrations to the posterior segment of the eye due to thepresence of the iris-lens barrier [31–35]. In contrast, transscleral/conjunctivaliontophoresis affords higher concentrations of ticarcillin, cefazolin, and gen-tamicin in the vitreous humor as a function of applied current density andduration [36]. Iontophoretic drug penetration heavily depends on the intensityand duration of applied currents, perhaps two of the major limitations ofthis technique from practical and commercial points of view. Recently, thistechnique was successfully applied for intraocular gene delivery of fluorescein-labeled anti-NOS oligonucleotides to the retina with an efficacy in the ratmodel of endotoxin-induced uveitis, suggesting that transscleral/conjunctivaliontophoresis facilitates the topical penetration of intact oligonucleotides intointraocular tissues [5].

13.3. An Overview of Conjunctival Disorders

Conjunctivitis, or “pink eye,” is an inflammation of the conjunctiva and prob-ably the most widespread form of ailment in this tissue. If left untreated,it can have serious consequences on vision and overall health. There aremany forms of conjunctivitis, and symptoms can range from itchy, burning,or teary eyes (allergic conjunctivitis) to more severe cases of bacterial andviral conjunctivitis (commonly known as “pink eye”). Each type requires


different treatment regimen and is highly contagious except for allergic con-junctivitis. Because of the similarity of the symptoms, only medical doc-tors trained well in ophthalmology can definitively distinguish between var-ious types of conjunctivitis and could determine the appropriate course oftherapy [37].

A number of ocular surface disorders collectively termed as “Dry EyeSyndromes” have also been associated with the conjunctiva. For example, adeficiency and/or imbalance in compositions of the tear film is often foundon the ocular surface during keratoconjunctivitis sicca. Since the conjunctivaplays a direct role in the maintenance of the tear fluid stability via secretionof mucin [1] by its resident goblet cells [4] and basal fluid secretion drivenby electro-osmotic gradients across the tissue [3], the conjunctiva is a welldeserved, but not intensively studied, target of interest in research efforts aimedagainst combating Dry Eye Syndromes.

Anatomical characteristics of disease in conjunctival inflammatory condi-tions have been reported. In contrast to the bacterial form of conjunctivitis, viralconjunctivitis shows a clear, watery discharge. The affected eye is irritated andred with a swollen eyelid. This condition is also highly contagious and whileonset is in one eye it can quickly infect the other eye. There are a number ofviruses that can trigger this form of conjunctivitis. Some viral infection is a rel-atively mild form that clears up on its own, while other viruses produce a moresevere form of the infection, such as herpes simplex conjunctivitis frequentlyseen in children or herpes zoster conjunctivitis which can lead to serious eyedamage. Adenovirus (Ad) is the most frequent viral cause of epidemic ocularinfections. Ocular Ad infection may lead to disseminated disease, which can befatal in children or in individuals with acquired immunodeficiencies. A wealthof knowledge about the relationship (that can be applied to improve treatment)between Ad and human ocular tissues is made possible with the developmentof cotton rat and rabbit models, Ad-induced pulmonary and ocular infections,together with the availability of human Ad mutants [38–40]. The adenovirustype 5 (Ad5) rabbit ocular infection model has been established for evalua-tion of various antiviral and anti-inflammatory agents in Ad5-induced oculardisease treatment [38].

13.4. Models for Studying ConjunctivalTransport Properties

13.4.1. Excised Conjunctival Tissues

The Ussing chamber technique, originally engineered to study transepithelialion transport, has been optimized to study drug transport and electrophysiologyof excised mammalian conjunctivas. This technique utilizes small tissue sheetsthat are mounted between two compartments of mucosal and serosal aspectsof the isolated conjunctival tissue, which are bathed with a physiologicalbuffer that is continuously bubbled with a stream of gas (compressed air or airbalanced with 5% CO2). In addition to providing sufficient oxygen tension formaintaining tissue viability, this configuration creates essential fluid circulationin each reservoir. A schematic diagram of Ussing chamber approaches is shownin Figure 13.2. Compounds of interest are added to either the mucosal or serosal


V

Automatic VoltageClamp Apparatus

A

Agar Bridges

CalomelElectrode

Ag/AgClElectrode Isolated

ConjunctivalTiussue

Bathing BufferBR/BRS, bubblingwith 5%CO2 in air

3M KCl

Figure 13.2 A schematic diagram of excised conjunctival tissues (shown) orTranswell-grown, primary cultured rabbit conjunctival epithelial cell layers mountedin a modified Ussing-type chamber. Two calomel electrodes for measurements oftranstissue voltages and two Ag/AgCl electrodes for passing electrical currents acrossthe conjunctival epithelial barrier are shown. This represents a four-electrode setupwhere one set of voltage and current electrodes connect via agar (containing 1–3 MKCl) bridges to mucosal (or apical) fluid and the other to serosal (or basolateral) fluid(See also color insert).

side of the excised conjunctiva (mounted in the chamber) to estimate fluxes inthe absorptive or secretory directions, respectively. Viability of isolated con-junctival tissues mounted in the Ussing chambers can be monitored continu-ously throughout transport studies, enabling the measurement of subtle changesdue to experimental agents (e.g., certain drugs under test) over those affordedby the vehicle alone in control tissues. Validation studies have demonstratedmeasurements of active transport properties of excised conjunctivas for at least4 h. Concurrent studies of paracellular transport markers (e.g., D-mannitol)are usually carried out to ascertain the integrity of the excised conjunctivaunder investigation. A separate study using morphological investigation oftissue ultrastructure for duration of experiments may provide some useful infor-mation. Monitoring tissue viability and integrity during the transport experi-ments is achieved by measuring transepithelial potential difference (PD) andresistance using the automatic voltage clamp unit. Other advantages of usingUssing chamber techniques for conjunctival tissue transport studies include theadded feasibility of looking at largely unknown mechanisms underlying oculardrug metabolism, regional absorption differences (i.e., bulbar, fornicial, orpalpebral conjunctiva segments), and relatively small amounts of drug neededto perform flux estimates, with analytically cleaner samples being collectible.While dissection of the tissue requires some practices that may be technicallytaxing at first, the conjunctival Ussing chamber model of excised tissues andprimary cultured conjunctival models (vide infra) are critical and rewardingtools for studies classifying compounds used in ophthalmic drug delivery. Inthis subsection, we provide a brief synopsis of Ussing chamber techniques.


Conjunctival short-circuit current, termed Isc, describes the cumulative sum ofall active, energy-driven, and electrogenic transport processes occurring acrossthe conjunctiva. The Ussing chamber apparatus can impose an experimentalcondition called “zero voltage clamp”, where the electrical PD developedacross the conjunctival tissue is forced to zero (i.e., offset to zero) by imposingan electrical PD of the same magnitude but opposite sign. The resultant currentflowing across the tissue is termed “short-circuit current” (Isc), bringing downthe spontaneously developed PD to zero (thus the name short-circuiting isused to describe the procedure). Under short-circuited conditions, there are noelectrical gradient across the tissue and by using the same Ringer’s solutionor culture medium on both sides of the barrier, the chemical gradients arealso zero. Thus, asymmetric fluxes of ions or solutes measured under suchzero gradient conditions across the barrier have to be energized by the barrieritself (e.g., via utilization of cell energy in the form of ATP consumption; e.g.,sodium pumps and/or vesicular transport dependent on cytoskeletal network-based movements of cell organelles and so forth). Surgically excised pigmentedrabbit conjunctival tissues display a tear side negative PD of 15.5 ± 1.5mV (mean ± S.E.M. of 70–80 determinations) when mounted in a modifiedUssing-type chamber [6–8, 41]. The voltage detection apparatus is composedof two matched calomel electrodes positioned very closely to the tissue sur-faces. A pair of Ag/AgCl electrodes is used to send and receive electrical cur-rents (that are generated by imposing an electrical PD of the same magnitudewith opposite sign of the observed PD), which are positioned away from thetissue surfaces in order to ensure uniform current density across the tissueand short the bath-tissue-bath circuit (Figure 13.2). Overall tissue resistance(transepithelial electrical resistance, TEER) can then be estimated by takingthe ratio between the observed PD and Isc, or TEER = PD/Isc (which describesthe Ohm’s law). Conjunctival TEER ranges from 0.75 to 1.5 k�·cm2 [6, 7].

A variety of automatic voltage clamp devices with special modificationshave been extensively utilized in electrophysiological studies of Isc in sev-eral ocular tissues including the amphibian corneal epithelium [42] andhuman fetal retinal pigment epithelium [43, 44], as well as non-ocular tissueslike the rat tracheal epithelium [45]. A strong temperature dependency andinhibitory effect of serosally instilled ouabain on the rabbit conjunctival Iscare characteristic of active ion transport driven by Na+/K+-ATPases in theconjunctiva [6, 7].

Utilizing healthy conjunctival tissues under various ex vivo configurationshas allowed for mechanistic studies of potential drug transport routes. More-over, it is just as feasible to surgically isolate conjunctivas from ocular diseasemodels for transport studies under pathological conditions with some limita-tions. Ocular infection models have been established using competent humanAd and thoroughly characterized in pigmented and albino rabbits [38, 39]. Ascompared to excised conjunctival tissues in health or disease, in vitro cultiva-tions of respective epithelial cells on permeable matrices under primary cultureconditions allow further investigative control on many additional dimensions.Studies of the conjunctival epithelial barrier alone, i.e., excluding the partic-ipation of systemic and lymphatic routes, provide a unique opportunity fora greater throughput in simultaneous transport investigations that consumeminimal animal resources with lowest data variability [26].


PIGMENTEDMALE

LIQUID COVEREDCONDITION

CULTURED CELLS AREPLACED UNDERAIR-INTERFACE

CONDITION FROM DAY 4

AIR INTERFACECONDITION

COATED FILTERSCULTURE MEDIUM

RESUSPEND CELLS INGROWTH MEDIUM

SEED ON TRANSWELLS®

40 µm CELLSTRAINER

FILTRATION

WASHCELLS

EXCISE EYEBALLFROM SOCKET &SEPARATE THECONJUNCTIVA

TREATMENT WITH APROTEASE SOLUTION

HARVESTCELLS

TREATMENT W

ITH

A DNase SOLUTION

SACRIFICE

Figure 13.3 A flowchart illustrating various steps for the preparation and maintenanceof primary rabbit conjunctival epithelial cell layers, cultured under liquid-covered andair-interfaced conditions (See also color insert).

13.4.2. Primary Culture Models of Conjunctival Epithelial Cell Layers

Cell cultures are a useful, high throughput means for evaluating drug transportmechanisms, impact of formulations, and physiological factors influencingabsorption across biological barriers (e.g., epithelia). Functional primary cul-tures of RCEC under conventional liquid-covered or advanced air-interfacedculture (AIC) conditions are available (Figure 13.3) [26, 46]. Conjunctivalepithelial cell cultures exhibit morphological as well as functional similaritiesto the tissue in vitro or in vivo. Cultured cells form functional tight junc-tions and express various enzymes (e.g., esterases, phosphodiesterases, andGSH metabolizing enzymes) and transporters (for amino acids, dipeptides, andnucleosides), similarly to those found in tissues in vitro or in vivo. Primary iso-lates of conjunctival epithelial cells achieve differentiated phenotypes and formmultilayer of cells, displaying epithelial characteristics with isles of mucin-secreting goblet cell populations. Culture time-dependent organization of tightjunctions, development of cell polarity, and expression of various transportmechanisms have been reported [26, 46]. Test compounds are typically addedto the apical side of the multilayer of cultured cells, while the cumulativeappearance in the basolateral compartment is measured using appropriate mea-surement schemes (e.g., radioactivity assays or fluorescence determinationsdepending on the tags on the drugs of interest). Evaluation of in vitro sys-tem integrity is performed through measuring electrical resistance across themultilayered cells cultured on permeable supports (e.g., TranswellTM systems)using specially engineered voltage-sensing and current-passing electrodes,attached to a portable voltohmeter (e.g., MilliCell device, Millipore Corp.,Billerica, MA, or EVOM device, World Precision Instruments, Sarasota, FL).


An alternative method for assessing cell layer integrity is through the useof hydrophilic paracellular transport markers (e.g., radiolabeled D-mannitolor fluorescein-Na+), which passively traverse cells by the paracellular route.Small amounts of compound required for in vitro conjunctival cell culturetransport experiments make this approach well suited for screening purposes.Relative absorption index of a series of pharmacologically active moleculescan be ranked against known markers for the identification of candidates withpotential absorption problems, which is a reliable tool to select drug candidateswith optimal characteristics.

13.4.3. Conjunctival Disease Models

Understanding differences in drug transport and metabolism in diseased con-junctival tissue is also important. A number of critical and physiological char-acteristics of conjunctival epithelial cells are altered in inflammatory states.An Ad5 infection model of the conjunctiva has been developed for utilityin studying modulations of transport mechanisms in health and disease [38].This virus-inoculated model has been adapted for tissue studies in the Ussingchamber configuration, as well as for primary cultures of conjunctival epithelialcells. The Ad5 infection model is a pertinent and critical tool for understandingthe ophthalmic drug delivery paradigm across the conjunctival tissue in (healthand) disease. Detailed protocols for this and other related subjects describedabove are included as appendix to this chapter.

13.5. Summary and Conclusions

The conjunctival tissue covers ∼80% of the ocular surface in higher mam-malian species and offers a unique route of delivering drugs locally to theeye. Paracellular, transcellular, active, and endocytic routes of drug absorp-tion play a key role in topical ocular delivery via the conjunctiva. Transscle-ral/conjunctival iontophoresis offers an alternative noninvasive topical deliverystrategy for charged drugs including macromolecular biologics. Lastly, thesubconjunctival route of drug delivery provides critical space and capacity forminimally invasive, safe, and sustained exposure of therapeutics to intraocularcompartments. The conjunctiva plays an important role in the etiology of anumber of ocular surface disorders as potential drug targets and an absorptionconduit. Multiple methods (see details in appendix) are available to study thepreclinical biopharmaceutics of the conjunctiva, facilitating physiological traitsof the conjunctiva, and improving the design of ocular drug delivery strategiesto the eye in health and disease.

Acknowledgments: Supported by grants HL38658 (KJK), HL64365 (KJK andVHLL), EY12356 (VHLL), and the Hastings Foundation. HJG thanks theAFPE, PhRMA, and USC Town and Gown. The authors appreciate contribu-tions by scientists who have worked on conjunctival transport in collaborationor directly in our laboratories (in the alphabetical order of last names): Drs P.Ashton, S. K. Basu, E. Hayakawa, Y. Horibe, K. I. Hosoya, S. D. Kashi, U. B.Kompella, A.A. Kulkarni, M. A. Narawane, M. G. Qaddoumi, J. R. Robinson,P. Saha, M. H. Shiue, R. E. Stratford Jr., L. Sun, H. Ueda, W. Wang, and J. J.Yang.


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14. Y. Horibe, K. Hosoya, K. J. Kim, T. Ogiso, and V. H. Lee. Polar solute transportacross the pigmented rabbit conjunctiva: size dependence and the influence of 8-bromo cyclic adenosine monophosphate. Pharm Res 14:1246–1251 (1997)

15. E. Hayakawa, D. S. Chien, K. Inagaki, A. Yamamoto, W. Wang, and V. H. Lee.Conjunctival penetration of insulin and peptide drugs in the albino rabbit. PharmRes 9:769–775 (1992)

16. R. E. Stratford, Jr. and V. H. Lee. Ocular aminopeptidase activity and distributionin the albino rabbit. Curr Eye Res 4:995–999 (1985)

17. R. E. Stratford, Jr., L. W. Carson, S. Dodda-Kashi, and V. H. Lee. Systemic absorp-tion of ocularly administered enkephalinamide and inulin in the albino rabbit:extent, pathways, and vehicle effects. J Pharm Sci 77:838–842 (1988)

18. L. Sun, S. K. Basu, K. J. Kim, and V. H. Lee. Arginine vasopressin transportand metabolism in the pigmented rabbit conjunctiva. Eur J Pharm Sci 6:47–52(1998)

19. M. G. Qaddoumi, H. J. Gukasyan, J. Davda, V. Labhasetwar, K. J. Kim, and V.H. Lee. Clathrin and caveolin-1 expression in primary pigmented rabbit conjunc-tival epithelial cells: role in PLGA nanoparticle endocytosis. Mol Vis 9:559–568(2003)


20. M. G. Qaddoumi, H. Ueda, J. Yang, J. Davda, V. Labhasetwar, and V. H. Lee.The characteristics and mechanisms of uptake of PLGA nanoparticles in rabbitconjunctival epithelial cell layers. Pharm Res 21:641–648 (2004)

21. K. Hosoya, Y. Horibe, K. J. Kim, and V. H. Lee. Na+-dependent L-argininetransport in the pigmented rabbit conjunctiva. Exp Eye Res 65:547–553 (1997)

22. K. I. Hosoya, Y. Horibe, K. J. Kim, and V. H. Lee. Carrier-mediated transportof NG-nitro-L-arginine, a nitric oxide synthase inhibitor, in the pigmented rabbitconjunctiva. J Pharmacol Exp Ther 285:223–227 (1998)

23. T. Hatanaka, M. Haramura, Y. J. Fei, S. Miyauchi, C. C. Bridges, P. S. Ganapathy,S. B. Smith, V. Ganapathy, and M. E. Ganapathy. Transport of amino acid-basedprodrugs by the Na+- and Cl−-coupled amino acid transporter ATB0,+ and expres-sion of the transporter in tissues amenable for drug delivery. J Pharmacol Exp Ther308:1138–1147 (2004)

24. B. S. Anand and A. K. Mitra. Mechanism of corneal permeation of L-valyl esterof acyclovir: targeting the oligopeptide transporter on the rabbit cornea. Pharm Res19:1194–1202 (2002)

25. S. K. Basu, I. S. Haworth, M. B. Bolger, and V. H. Lee. Proton-driven dipeptideuptake in primary cultured rabbit conjunctival epithelial cells. Invest OphthalmolVis Sci 39:2365–2373 (1998)

26. J. J. Yang, H. Ueda, K. Kim, and V. H. Lee. Meeting future challenges in topicalocular drug delivery: development of an air-interfaced primary culture of rabbitconjunctival epithelial cells on a permeable support for drug transport studies. JControl Release 65:1–11 (2000)

27. K. Hosoya, Y. Horibe, K. J.Kim, and V. H. Lee. Nucleoside transport mechanismsin the pigmented rabbit conjunctiva. Invest Ophthalmol Vis Sci 39:372–377 (1998)

28. H. Z. Bu, H. J. Gukasyan, L. Goulet, X. J. Lou, C. Xiang, and T. Koudriakova. Ocu-lar disposition, pharmacokinetics, efficacy and safety of nanoparticle-formulatedophthalmic drugs. Curr Drug Metab 8:91–107 (2007)

29. U. B. Kompella, N. Bandi, and S. P. Ayalasomayajula. Subconjunctival nano- andmicroparticles sustain retinal delivery of budesonide, a corticosteroid capable ofinhibiting VEGF expression. Invest Ophthalmol Vis Sci 44:1192–1201 (2003)

30. J. A. Gilbert, A. E. Simpson, D. E. Rudnick, D. H. Geroski, T. M. Aaberg, Jr.,and H. F. Edelhauser. Transscleral permeability and intraocular concentrations ofcisplatin from a collagen matrix. J Control Release 89:409–417 (2003)

31. R. E. Grossman, D. F. Chu, and D. A. Lee. Regional ocular gentamicin levels aftertranscorneal and transscleral iontophoresis. Invest Ophthalmol Vis Sci 31:909–916(1990)

32. D. Maurice. Iontophoresis and transcorneal penetration of tobramycin. Invest Oph-thalmol Vis Sci 30:1181–1182 (1989)

33. D. S. Rootman, J. A. Jantzen, J. R. Gonzalez, M. J. Fischer, R. Beuerman, and J.M. Hill. Pharmacokinetics and safety of transcorneal iontophoresis of tobramycinin the rabbit. Invest Ophthalmol Vis Sci 29:1397–1401 (1988)

34. B. S. Kwon, L. P. Gangarosa, N. H. Park, D. S. Hull, E. Fineberg, C. Wiggins,and J. M. Hill. Effect of iontophoretic and topical application of antiviral agentsin treatment of experimental HSV-1 keratitis in rabbits. Invest Ophthalmol Vis Sci18:984–988 (1979)

35. J. M. Hill, N. H. Park, L. P. Gangarosa, D. S. Hull, C. L. Tuggle, K. Bowman,and K. Green. Iontophoresis of vidarabine monophosphate into rabbit eyes. InvestOphthalmol Vis Sci 17:473–476 (1978)

36. M. Barza, C. Peckman, and J. Baum. Transscleral iontophoresis of cefazolin,ticarcillin, and gentamicin in the rabbit. Ophthalmology 93:133–139 (1986)

37. C. G. Begley, B. Caffery, R. L. Chalmers, and G. L. Mitchell. Use of the dry eyequestionnaire to measure symptoms of ocular irritation in patients with aqueoustear deficient dry eye. Cornea 21:664–670 (2002)


38. Y. J. Gordon, E. Romanowski, and T. Araullo-Cruz. An ocular model of adenovirustype 5 infection in the NZ rabbit. Invest Ophthalmol Vis Sci 33:574–580 (1992)

39. M. D. Trousdale, R. Nobrega, D. Stevenson, T. Nakamura, P. M. dos Santos,L. LaBree, and P. J. McDonnell. Role of adenovirus type 5 early region 3 inthe pathogenesis of ocular disease and cell culture infection. Cornea 14:280–289(1995)

40. J. C. Tsai, G. Garlinghouse, P. J. McDonnell, and M. D. Trousdale. An experi-mental animal model of adenovirus-induced ocular disease. The cotton rat. ArchOphthalmol 110:1167–1170 (1992)

41. K. Hosoya, U. B. Kompella, K. J. Kim, and V. H. Lee. Contribution of Na+-glucosecotransport to the short-circuit current in the pigmented rabbit conjunctiva. CurrEye Res 15:447–451 (1996)

42. O. A. Candia and P. S. Reinach. Thermodynamic analysis of active sodium andpotassium transport in the frog corneal epithelium. Am J Physiol 242:F690-F698(1982)

43. R. H. Quinn and S. S. Miller. Ion transport mechanisms in native human retinalpigment epithelium. Invest Ophthalmol Vis Sci 33:3513–3527 (1992)

44. R. H. Quinn, J. N. Quong, and S. S. Miller. Adrenergic receptor activated iontransport in human fetal retinal pigment epithelium. Invest Ophthalmol Vis Sci42:255–264 (2001)

45. J. S. Jung, J. Y. Lee, S. O. Oh, P. G. Jang, H. R. Bae, Y. K. Kim, and S. H. Lee. Effectof t-butylhydroperoxide on chloride secretion in rat tracheal epithelia. PharmacolToxicol 82:236–242 (1998)

46. P. Saha, K. J. Kim, and V. H. Lee. A primary culture model of rabbit conjunctivalepithelial cells exhibiting tight barrier properties. Curr Eye Res 15:1163–1169(1996)

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The Conjunctival Barrier in Ocular Drug Delivery