Modern approaches to the ocular delivery of cyclosporine A

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Modern approaches to the ocular delivery of cyclosporine A Priyanka Agarwal and Ilva D. Rupenthal Buchanan Ocular Therapeutics Unit, Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, University of Auckland, New Zealand

Cyclosporine A (CsA) has long been the mainstay treatment for dry eye syndrome (DES), one of the most common disorders of the eye. However, the poor water solubility of CsA renders it difficult to formulate it into topical ocular dosage forms. Restasis1 is currently the only US Food and Drug Administration (FDA)-approved CsA formulation, while Ikervis1 has recently been launched in Europe, with both commonly associated with severe ocular discomfort. Therefore, several CsA formulations have been investigated with the aim to improve bioavailability while reducing adverse effects associated with the marketed formulations. In this review, we summarize recent advances in ocular CsA delivery that provide safer and more effective alternatives for the management of DES and other ocular inflammatory conditions. Introduction CsA is a metabolite of the fungi Tolypocladium inflatum and Beauveria nevus that was initially suggested for use as an antifungal agent. Its immunosuppressive activity soon became evident and, because of the reduced incidence of associated myelotoxicty, CsA eventually became the mainstay treatment after organ transplantation [1,2]. Systemic CsA is still used, although to a lesser extent, to treat several other autoimmune diseases, including those with eye involvement [3]. CsA is the preferred immunomodulatory agent for topical treatment of several immunemediated ocular surface disorders [4], and its prevalence in the treatment of these ocular disorders is second only to corticosteroids, whose adverse effects are well documented [5]. CsA therapy has been approved for the treatment of keratoconjunctivitis sicca (KCS), more commonly known as dry eye syndrome (DES). It is also frequently used off-label to treat several other ophthalmic conditions, such as posterior blepharitis [6,7], ocular rosacea [8,9], vernal keratoconjunctivitis [10–14], atopic keratoconjunctivitis [15–17], acute corneal graft rejection [18], and conjunctival graft versus host disease [19–21]. DES is one of the most prevalent ocular surface disorders, and is usually characterized by increased evaporation or decreased production of tear fluid, resulting in damage to the interpalpebral ocular surface and moderate to severe discomfort [22]. Symptoms of DES have been reported in approximately one out of seven individuals above the age of 48, with its prevalence nearly doubling after 59 years of age [23–25]. A recent study estimated that nearly 20% of

Priyanka Agarwal is currently pursuing a doctorate within the Buchanan Ocular Therapeutics Unit, Department of Ophthalmology, University of Auckland, and is investigating novel cyclosporine A (CsA) formulations for topical administration. She completed her BPharm at the University of Mumbai, India, and subsequently obtained a Postgraduate Diploma in Health Sciences from the School of Pharmacy at the University of Auckland. Priyanka has extensive industrial research experience in formulation development and pharmacokinetics and worked as a Veterinary Formulation Development Scientist for several years. During her time in industry, she primarily worked on the development of new platforms for safe drug delivery to animals and she is currently one of the inventors on a series of patents filed in this field. Ilva Rupenthal is a senior lecturer in the Department of Ophthalmology, New Zealand National Eye Center, University of Auckland, and the inaugural Director of the Buchanan Ocular Therapeutics Unit, which aims to translate ocular therapeutic-related scientific research into the clinical setting, whether pharmaceutical, cell, or technology based. Her current research, funded by a Sir Charles Hercus Health Research Fellowship from the New Zealand Health Research Council, focuses mainly on the development of stimuli-response devices, with projects investigating ocular implants responsive to light or a small electrical current. Moreover, Dr Rupenthal’s group is developing tailored controlled delivery systems that specifically target the drug to the site of action with projects around dry eye, optic neuropathy, diabetic retinopathy, and age-related macular degeneration treatment.

Corresponding author: Rupenthal, I.D. ([email protected]) 1359-6446/ß 2016 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drudis.2016.04.002

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Given that cyclosporine (CsA) is poorly water soluble and currently marketed products are not well tolerated, novel approaches for safe and efficient CsA delivery to the eye are of great interest.

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hospitalized patients above the age of 50 had DES, with old age and illiteracy being major predictors of the disorder [26]. On the recommendation of the International Dry Eye Workshop in 2007, DES was classified as a multifactorial disease of the tears and ocular surface that can be triggered by a variety of underlying causes. However, recently, there has been increasing evidence that inflammation has a key role in the manifestation of DES. Ocular surface abnormalities, such as the appearance of inflammatory cell intermediates in the lacrimal gland and an increase in immunerelated antigens and cytokines at the conjunctival epithelium, ¨ gren’s are commonly demonstrated by both autoimmune (Sjo ¨ gren’s syndrome)syndrome) and non-autoimmune (non-Sjo mediated DES [27–30], making treatment of the underlying cytokine–receptor-mediated inflammatory processes the primary ‘causative therapeutic approach’ [31,32]. A combination of symptomatic therapy, which includes modification of the ocular environment (by increasing humidity, occlusion of lacrimal canaliculi, or simulation of tears), and pathogenic treatments, including the use of antibacterial and anti-inflammatory agents (corticosteroids, antihistamines, tetracyclines, and CsA), is currently recommended for DES therapy [32]. CsA is the anti-inflammatory agent of choice for the treatment of DES, because it can be used long term without adverse effects commonly associated with other anti-inflammatory agents, such as steroids [5]. Furthermore, unlike corticosteroids, the activity of CsA results from specific and reversible action on T cells, making it safe for prolonged use. For example, corneal epitheliopathy and eyelid maceration associated with long-term CsA therapy were found to be reversible with complete cessation on discontinuation of the drug [33]. The immunomodulatory activity of CsA helps in reducing the inflammation associated with subconjunctival and lacrimal glands, resulting in increased goblet cell density and tear production [34,35]. CsA tends to bind with specific nuclear proteins that initiate the activation of T cells, thus preventing T cell production of inflammatory cytokines and disrupting the immune-mediated inflammatory response [31,36]. CsA is a hydrophobic molecule and, therefore, is difficult to formulate into conventional topical ocular delivery systems. Significant research has been performed over recent years to develop safe and effective ocular delivery systems for CsA. In this review, we highlight recent efforts to improve the ophthalmic delivery of CsA in terms of its bioavailability and ocular tolerability while reducing adverse effects.

Conventional CsA formulations Systemic CsA is generally not considered for the treatment of ocular pathologies because of severe systemic adverse effects, such as nephrotoxicity and hypertension [2,37], although significant concentrations of CsA have been reported in tears and lacrimal glands after oral administration [38]. Thus, the topical route is generally preferred because, as well as reducing systemic adverse effects, it also helps to achieve improved bioavailability and specific targeting to the ocular tissues [39,40]. Dose-ranging randomized clinical trials have shown that topically applied CsA is effective at concentrations between 0.05 and 0.1% (w/v). No additional benefits were observed at higher concentrations; hence, clinical trials are generally recommended at a maximum concentration of 0.1% [4,41]. However, because CsA is a 2

Drug Discovery Today  Volume 00, Number 00  April 2016

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Drug Discovery Today

FIGURE 1

Molecular structure of cyclosporine A (CsA), a cyclic undecapeptide with very low aqueous solubility.

neutrally charged and hydrophobic molecule (Fig. 1) with an aqueous solubility of less than 10 mg/ml (0.001%) at physiological temperature [42], formulation of aqueous eye drops at these concentrations is difficult. Attempts have been made to improve the aqueous solubility of CsA using surfactants and/or penetration enhancers, such as macrogolglycerol ricinoleate (Cremophor EL1) and benzalkonium chloride, with the latter also commonly used as a preservative in ocular formulations [43]. Although these excipients can improve the solubility and penetration of CsA, their use is limited by their high irritation potential because these molecules typically function by compromising the integrity of the ocular tissues [44–46]. CsA solutions in vegetable oils were considered as the next best alternative for topical administration to the eye and concentrations as high as 2% could be achieved [47,48]. Despite their poor absorption, oily CsA solutions have demonstrated success in the treatment of DES by improving tear production and inducing regression of corneal neovascularization in a canine model [49–54]. Similar results were also observed in humans after topical application of 2% CsA in olive oil [55,56]. However, one of the major limitations of vegetable oils in ophthalmic preparations is the increased incidence of ocular toxicity after frequent use, with blurring of vision also commonly observed because of the high viscosity of the carrier oils. BenEzra et al. [38,57,58] showed that penetration of CsA from olive oil drops was negligible in normal or slightly inflamed eyes, but increased significantly over time because the corneal barrier was compromised as a result of toxic effects of the oily vehicle. Thus, the use of topical CsA oily drops for the management of ophthalmic conditions has largely been discontinued. CsA ointments [59–61] and oil-in-water (o/w) emulsions [62] have also been used to reduce the toxicity associated with oily solutions, although they have not been able to eliminate it completely. Currently, Restasis1 (Allergan) is the only CsA formulation approved by the FDA for DES therapy in humans. It is a 0.05% CsA emulsion of castor oil in water and is often associated with severe adverse effects, such as ocular burning (most common), conjunctival hyperemia, discharge, epiphora, eye pain,

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Recent approaches in CsA delivery Over the past few years, several novel strategies have been suggested for delivering CsA to the ocular tissues, with topical, episcleral, subconjunctival, and intravitreal routes being investigated. Of these, topical administration remains the most preferred route for the treatment of DES, because it is non-invasive, painless, and convenient. For the purpose of this review, the key approaches to improving ocular drug delivery of CsA have been classified into three major areas of research: (i) chemical modification of the drug; (ii) novel application of excipients; and (iii) novel ophthalmic dosage forms.

Chemical modification of the drug Reversible chemical modification of CsA to obtain prodrugs with improved aqueous solubility was first suggested by Hammel et al. [68] when they coupled CsA with diketopiperazine to synthesize dipeptide esters with improved oral bioavailability. Monomethoxy(polyethyleneglycol) derivatives of CsA have also been suggested for improving solubility and absorption of CsA in oral preparations. However, esterification of the free hydroxyl group of CsA with a solubilizing moiety is the generally accepted approach for prodrug synthesis for ocular applications. Lallemand et al. [69,70] developed a series of amphiphilic acidic prodrug molecules (patented by DeBiopharm, Switzerland) having an approximately 25 000 times higher solubility than CsA in isotonic phosphate buffer solution (PBS) at pH 7. These prodrugs are quantitatively hydrolyzed in artificial tears to release CsA within 1 min. Prodrug conversion into the parent molecule was significantly faster in tear fluid than in a buffer at physiological pH, indicating that the hydrolysis is enzyme mediated. Aqueous formulations of these esterified CsA prodrugs were well tolerated and have shown

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FIGURE 2

Ocular distribution of cyclosporine A (CsA) in oil and CsA prodrug in water. Prodrug formulations significantly increased the amount of CsA absorbed by, and accumulated in, the cornea and conjunctiva; however, no improvement was observed in the penetration of CsA across the cornea. Source: Adapted from [75].

a significant improvement in basal tear production in DES [71,72]. Aqueous prodrug solutions have also been evaluated for their efficacy in the treatment of corneal graft rejection and it was found that prodrug eye drops applied five times a day were therapeutically equivalent to a 10 mg/kg/day intramuscular injection in rats [73]. Recent studies have shown that 2% prodrug solutions have a 200–500-fold higher conjunctival permeability than the conventional 2% CsA in oil formulation. Accumulation of CsA prodrug formulations in the cornea to form large tissue deposits that provide a sustained release effect over prolonged periods of time was also observed. However, prodrug formulations did not show much improvement in the permeability across the cornea and into the aqueous humor compared with the conventional CsA emulsion, probably because of the rapid conversion of the prodrug into CsA at the corneal surface (Fig. 2). This depot formation could have the added advantage of overall poor systemic absorption of prodrug formulations, reducing the incidence of systemic complications and immunosuppression. Prodrug formulations with CsA concentrations higher than 2% are currently under investigation for safety and toxicity [74,75].

Novel application of excipients Several new excipients have been introduced to improve CsA solubility in ocular formulations and enhance their bioavailability (Table 1).

Cyclodextrins The introduction of cyclodextrins (CDs) in ophthalmic drug delivery is relatively recent; however, they have rapidly gained popularity because of their pronounced benefits in drug solubilization and stabilization. CDs are cyclic oligosaccharides of six (a-CD), seven (b-CD), or eight (g-CD) glucopyranose units with a hydrophobic cavity in the center. In aqueous solutions, water from the hydrophobic cavity is displaced by hydrophobic molecules, resulting in the formation of large water-soluble complexes. CDs have been used on several occasions to prepare aqueous CsA

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Total CsA (ng/g)

foreign body sensation, pruritus, stinging, and visual disturbance [63]. Devecı et al. [64] recently showed that ocular irritation associated with Restasis usually decreases within 1 week of continuous therapy and significant resolution of DES can be observed at the 1-month follow-up. A veterinary ointment of 0.2% CsA (Optimmune1, Schering-Plough Animal Health) has also been approved for DES therapy in dogs and, although it has been shown to resolve the symptoms of DES, associated ocular toxicity, probably because of the oily base, is significant [65,66]. Ointments are generally more viscous and have a cloudy appearance and, thus, tend to blur the vision, which, in addition to their tolerability issues, reduces patient acceptability. Recently, a cationic nanoemulsion containing 0.1% CsA was launched in Europe for the treatment of severe DES under the brand name Ikervis1 (Santen). Unlike emulsions and ointments, this system does not cloud vision because of its low viscosity; however, adverse effects, such as stinging and pain, have frequently been reported [67]. The limitations of currently approved formulations leave tremendous scope for the development of improved ocular formulations for the safe and efficient delivery of CsA to the eye. The focus of new CsA technologies has predominantly been the improvement of solubility, transcorneal penetration, and precorneal residence time, with a simultaneous reduction in the frequency of dosing and irritation potential. Such CsA formulations could significantly improve patient comfort and compliance, thus increasing the quality of life of patients with DES.

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TABLE 1

Novel excipients used in ocular CsA delivery systems.

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Excipients

Dosage form

Advantages

Limitations

Refs

Cyclodextrins

Aqueous eye drops

Solubilize CsA in aqueous formulations; show improved bioavailability and low toxicity at optimized concentrations

Cannot be used in concentrated solutions; ocular toxicity can be observed at higher concentrations because of complexation of cellular components

[76–79]

Semifluorinated alkanes

Eye drops (CyclASolW)

Safe and well tolerated; solubilize hydrophobic drugs to form clear eye drops; improved penetration; no preservative required; lubricating nature

No sustained release effect

[80,81]

Cationic vectors (amines, chitosan, poly-L-lysine, Eudragit)

Emulsions, liposomes, nanoparticles

Improve precorneal residence time because of mucoadhesion

Amine vectors can cause stability problems; safety and toxicity is a concern because ocular irritation is usually observed with most cationic moieties

[95]

eye drops with significant improvement in transcorneal penetration and retention [76–79]. a-CDs have been found to be the most efficient in solubilizing CsA, because the cavity size is a good fit for the cyclic CsA molecule. When used to prepare concentrations below 0.025%, a-CDs improved CsA bioavailability without demonstrating any toxic effects in vivo [77,79].

Semi-fluorinated alkanes (SFAs) SFAs are a class of amphiphilic fluorinated compounds that are physically, chemically, and biologically inert and capable of dissolving several hydrophobic drugs. Although they have been used intraocularly as intravitreal tamponades for several years, their use in drug delivery to the front of the eye is relatively recent [80]. Given their established safety profile, excellent spreading properties, and ability to solubilize and stabilize several hydrophobic drugs, SFAs can be used as suitable carriers for ocular preparations. The added advantages of SFAs include their ability to form clear solutions that do not cause any blurring of vision or ocular discomfort while also providing lubrication to the ocular surface, which is particularly useful in DES treatment. Moreover, because these solutions are nonaqueous, they do not require any preservatives, surfactants, or pH modifiers, which are frequently implicated in ocular toxicity. Recent studies showed that SFA-based solutions containing 0.05% CsA were well tolerated and significantly increased CsA penetration across the cornea and into the aqueous humor when compared with Restasis, rendering SFAbased formulations promising candidates for the treatment of ocular inflammatory conditions [81]. This technology is currently registered as CyclASol1 (Novaliq GmbH, Germany) and recently conducted Phase I clinical trials have shown promising results [82], with the company now recruiting patients for a Phase II clinical trial [83].

Positively charged vectors One of the major objectives of topical CsA delivery has been to improve ocular bioavailability of the drug by increasing corneal penetration and precorneal residence time. Therefore, positively charged ocular formulations have received much interest because of their higher mucoadhesion and, thus, longer precorneal residence time. Studies performed by Daull et al. [84] showed that cationic emulsions have significantly greater bioavailability than Restasis, which is an anionic o/w emulsion. Safety profiles of 4

cationic and anionic emulsions were further compared and, while their tolerability was similar, only the cationic emulsion was able to maintain the normal healing rate of the human corneal epithelium in vitro and reduce inflammation in vivo [85]. Similar to most epithelia, corneal and conjunctival cells are negatively charged at physiological pH and, therefore, cations can adhere to and penetrate them more easily [86,87]. Their cell membranes are further coated with a layer of mucin containing negatively charged sialic acid groups, which develop electrostatic interactions with positively charged vectors and thus improve the precorneal residence time [88]. Stearylamine has been used extensively to impart a positive charge to liposomes [89–91] and ocular emulsions [92–94] to increase mucoadhesion and, thus, retention. Oleylamine is another cationic lipid that has been used for preparation of cationic ophthalmic emulsions [88]. However, a major disadvantage of these amines is their poor stability and compatibility with other excipients, while ocular tolerability of amines remains a significant concern. Hence, chitosan has emerged as the cationic agent of choice in ocular formulations. Chitosan is a biodegradable and biocompatible linear polysaccharide with several positively charged free amino groups that interact with mucin. There is some evidence that, in addition to the positive charge, the specific nature of chitosan might also be responsible for improving the uptake of drug molecules. Calvo et al. [95] showed that the bioavailability of nanocapsules coated with chitosan was at least twofold higher than for poly-L-lysine-coated nanocapsules. However, commercialization of chitosan formulations might be difficult, because raw materials of natural origin often demonstrate high batch-to-batch variability. Recently, several studies also reported that, despite its biodegradability, chitosan can display some toxicity, especially when administered as nanoparticles [96,97], while chitosan hydrogels of a certain molecular weight have also shown to initiate an inflammatory response and, thus, delay wound closure in a corneal scrape wound model [98]. Therefore, Eudragit1, a synthetic polymer frequently used for the preparation of enteric oral dosage forms, has been suggested as an alternative for ophthalmic formulations [99]. Eudragit is a cationic copolymer based on dimethylaminoethyl methacrylate, butyl methacrylate, and methyl methacrylate groups. Eudragit-based ocular colloidal formulations have shown good tolerability and

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TABLE 2

Drug delivery system

Advantages

Limitations

Refs

Micelles

Improved uptake into all layers of the cornea; sustained release over extended periods

Generally poor shelf life; irritation potential and toxicity can be significant

[103–106]

Form depots in cornea for sustained drug release over prolonged periods Improved stability and simple manufacturing procedure

Short half-life at the corneal surface results in poor transcorneal penetration Safety and irritation potential need to be assessed

[113,114,118]

Improved retention and uptake into cornea; reduced drug toxicity can be observed Improved uptake because of transport by transcellular pathway Improved bioavailability; improved shelf-life compared with nanoemulsions

Difficult to scale up; can show some long-term toxicity Generally poor stability and manufacturability Generally poor long-term stability and manufacturability

[143–145]

Increased precorneal residence time and sustained release Sustained release of the drug over prolonged periods

High burst release; blurring of vision can reduce patient compliance Discomfort and foreign body sensation; adverse effects because of poor oxygen permeability

[154,157,160]

Liposomes Liposomes Proliposomes Nanoparticles Nanospheres/nanocapsules Nanoemulsions Solid lipid nanoparticles Other In situ gelling systems Hydrogels

low incidence of ocular irritation [100–102], while decomposition of Eudragit is typically slow, providing controlled drug release with a low burst effect.

Novel ophthalmic dosage forms The field of ocular therapeutics has seen the development of several novel delivery systems in the form of micelles, liposomes, nanoparticles, in situ gelling systems, and hydrogels, which have also been evaluated for the delivery of CsA (Table 2 and Fig. 3).

[119]

[133,134] [142]

[164,165,171]

Micelles Micelles are self-assembling spherical colloidal systems that are frequently used for the solubilization of hydrophobic molecules. Typically, micelles are formed around a hydrophobic drug in aqueous solution because of the orientation of surfactant molecules to form a hydrophobic core enclosing the drug within a hydrophilic shell. N-Octyl chitosan has been used as a surfactant for preparation of CsA micelles for DES therapy [103]. CsA uptake from these micelles was found to be significantly higher than from

In situ gelling systems + Improved retention + Sustained release

- Blurring - Burst release

Hydrogels + Improved retention + Sustained release

- Patient discomfort - Poor oxygen permeability

Micelles + Improved absorption + Deeper penetration

- Surface toxicity - Poor stability

Liposomes + Corneal accumulation + Sustained release

- Poor scalability - Low entrapment

Nanoparticles + Improved penetration + Improved absorption

- Poor scalability - Poor stability Drug Discovery Today

FIGURE 3

Schematic representation of novel ophthalmic dosage forms used to improve the bioavailability of topically applied cyclosporine A (CsA). www.drugdiscoverytoday.com 5 Please cite this article in press as: Agarwal, P., Rupenthal, I.D. Modern approaches to the ocular delivery of cyclosporine A, Drug Discov Today (2016), http://dx.doi.org/10.1016/ j.drudis.2016.04.002

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Novel ophthalmic dosage forms for CsA delivery to the front of the eye.

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conventional CsA oil drops, and the bioavailability could be further increased by coating the micelles with carbopol to give them mucoadhesive properties. Penetration of micellar formulations can also be enhanced by reducing the size of the micelles. In a recent study using an optimized blend of hydrogenated castor oil40 and octoxynol-40, Cholkar et al. [104] prepared a clear aqueous nanomicellar solution containing 0.025–0.1% CsA with an average particle size of 22.4 nm. High corneal and conjunctival levels were observed with low ocular toxicity. One of the most significant findings of this study was that this formulation could also deliver CsA to the posterior segment of the eye, with as much as 53.7 ng/g of CsA recovered from the retina/choroid in vivo. Polyvinyl caprolactam–polyvinyl acetate–polyethylene glycol nanomicelles have also shown improved penetration of CsA along with excellent ocular tolerability and safety [105]. CsA uptake from these micelles is predicted to be an energy-dependent intracellular pathway, leading to improved efficacy in vivo. Recently, in vivo studies performed with nano-sized micellar systems based on methoxy poly(ethylene) glycol-hexyl substituted poly(lactide) (mPEGhexPLA) copolymers showed increased uptake and prolonged release of CsA because of better penetration into, and accumulation in, all layers of the cornea [106]. Significant advantages of this mPEGhexPLA nanomicellar system included the absence of ocular toxicity and a longer shelf life compared with conventional micelle systems [107,108]. Further improvement in shelf life by lyophilization of mPEGhexPLA micellar systems has also been suggested; however, long-term studies still need to be performed to verify this [109]. Poor long-term stability is the major drawback of most micelle systems. Therefore, in an attempt to overcome this limitation, CsA-loaded self-assembling micellar systems were prepared using two PEG-fatty alcohol ether-type surfactants [110]. The micellar formulation had good entrapment efficiency and showed a threefold increase in corneal CsA concentration compared with Restasis. However, the toxicity and irritation potential of this formulation still needs to be evaluated.

Liposomes Over the past few decades, liposomes have been used extensively for drug solubilization and targeting. Liposomes are membranelike spherical vesicles comprising phospholipid bilayers that can efficiently solubilize both hydrophilic and hydrophobic drugs [111]. Typically, liposomal CsA formulations tend to accumulate in the cornea to form deposits for sustained delivery over a prolonged period of time [112,113]. CsA concentrations in aqueous and vitreous humor after treatment with nanoliposomes prepared by solvent evaporation were low but comparable with Restasis [113], and could be attributed to the short half-life of the liposomes on the corneal surface, resulting in release of free CsA from the liposomes before they could penetrate all the layers of the cornea. As discussed above, liposomes modified with a positive surface charge can improve precorneal residence and transcorneal penetration, resulting in improved bioavailability; however, the improvement in drug absorption is usually accompanied by a simultaneous increase in toxicity and inflammation [114,115]. Studies performed using a series of chitosan formulations have shown that the precorneal retention time remains unchanged on changing the molecular weight and/or concentration of chitosan used, suggesting that the bioadhesive properties of chitosan result 6

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from a saturable interaction with mucin [116] and, thus, concentrations could be optimized to achieve maximal mucoadhesion with minimal toxicity. Li et al. [117] prepared liposomes coated with low-molecular-weight chitosan and observed a significant improvement in the ocular bioavailability of CsA. The number of free positively charged groups available in low-molecular-weight chitosan is considerably reduced, resulting in better ocular tolerability of these formulations. Despite their many benefits, the fast degradation on the corneal surface, poor entrapment efficacy, and difficulty in manufacturing sterile formulations at a large scale are disadvantageous to the use of liposomal formulations. To counter these problems, Karn et al. [118] developed CsA-loaded multilamellar liposomes from egg lecithin and phosphatidyl choline from soybean using supercritical fluid of carbon dioxide as the antisolvent. Liposomes, thus prepared, showed better stability on the corneal surface, had an improved shelf life, and were easy to manufacture on a larger scale. Considerable improvements were also seen in the precorneal residence time, corneal absorption, and tolerance in comparison to Restasis [112]. Recently, the same method was also used to prepare proliposomes [119], which are dry free-flowing powders that assemble spontaneously in an aqueous medium to form liposomes. These proliposomes had excellent stability and were easy to scale up. However, some additional studies might be required to establish their efficacy in ocular CsA delivery.

Nanoparticles A large body of research has also been dedicated to the development of nanoparticle-based systems for targeted CsA delivery to the eye. Nanoparticles are submicron particles ranging from 10 to 1000 nm in size that can have drug molecules dissolved, adsorbed, or entrapped in the nanoparticle matrix. Nanoparticles have been shown to accelerate drug penetration and significantly improve corneal absorption of drugs, primarily by the transcellular pathway [120–123]. They have also been reported to decrease the incidence of ocular irritation and toxicity because of their small particle size [124,125]. Moreover, because they are similar to aqueous eye drops in appearance and viscosity, nanoparticle formulations are easy to handle and ensure convenient ocular application [126–128]. However, despite their advantages, products based on nanotechnology rarely reach the market [129] as a result of technical issues involved in scale up and manufacturing, use of new excipients or nonpharmacopoeial solvents [130], as well as the relatively poor stability of these colloidal systems [131]. CsA nanosuspensions have been prepared using zirconia beads in a water/polyvinyl alcohol system and were found to show significantly lower irritation compared with Restasis [132]. However, biodegradable polymers, such as poly-D,L-lactide-co-glycolide (PLGA) and poly-e-caprolactone-co-lactide (PCL), are usually preferred for ophthalmic applications because of their established safety profile. PLGA nanoparticles prepared by solvent evaporation have shown a 11–16% drug-loading capacity and have been used for sustained release of CsA for up to 65 days [133]. It was found that coating these nanoparticles with a surface-modifying polymer, such as PEG, could influence the penetration behavior of CsA across the cornea. PCL nanoparticles have also been used for ocular CsA delivery in conjugation with penetration-enhancing surfactants, such as benzalkonium chloride [134]. Although the bioavailability from these nanospheres was 10–15 times higher than

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from castor oil, their irritation potential is expected to be higher because of the presence of benzalkonium chloride. To increase the mucoadhesive properties of CsA nanoparticles, Liu et al. [135] recently prepared PLGA and dextran nanoparticles surface modified with phenylboronic acid. Improved safety and drug release from this formulation were observed over prolonged periods, with a once-a-week dose being suitable for reducing inflammation and promoting corneal surface healing. Cationic nanoparticles, showing improved retention and absorption on the corneal surface, were also prepared by coating PLGA particles with Eudragit RL, which is commonly used for the preparation of controlled-release oral dosage forms [99,136]. High cellular uptake and tear fluid concentrations (366.3 ng/g) of CsA were observed using this formulation. However, chitosan is more commonly used for introducing a positive charge to nanoparticles to increase mucoadhesion and, thus, ocular bioavailability [95]. Cationic chitosan nanoparticles have shown a significant increase in precorneal retention and drug uptake, resulting in sustained delivery of CsA to the aqueous and vitreous humor over 72 h [137,138]. However, it has been demonstrated that chitosan-coated nanocapsules typically do not traverse the entire epithelium, but tend to be retained in the superficial cell layers only [120]. One of the major challenges to the commercialization of such nanoparticles is their relatively poor stability in colloidal solutions. To counter this problem, Luschmann et al. [139] developed phase-sensitive in situ-precipitating nanosuspensions using PEG and Solutol1 as the non-ionic surfactant and co-surfactant, respectively. These formulations were shelf-stable clear liquids that precipitated in situ in the presence of moisture to form nanosuspensions and showed a sixfold increase in uptake of CsA compared with Restasis [140]. However, the application of this system in DES therapy is questionable, because corneal moisture levels are usually low to negligible in severe DES and, therefore, CsA would be unlikely to precipitate completely. The potential of nanoemulsions in ocular drug delivery is evident, because, by enclosing the drug in a small lipid droplet, its penetration by the transcellular pathway can be enhanced. Positively charged ocular emulsions have been reported to significantly improve the spreading coefficient and contact angle on the corneal epithelium [93,141], making them an attractive alternative for ocular drug delivery. However, a major limitation of conventional emulsions is their short shelf life and poor manufacturability. To overcome these limitations, Santen has developed Novasorb1, a stable sterile cationic nanoemulsion using benzalkonium chloride and cetalkonium chloride as the cationic vectors [142]. As discussed above, benzalkonium chloride, in addition to being a positive vector and preservative, also serves as an ocular penetration enhancer, but has a rather high irritation potential. However, it has been suggested that, by formulating benzalkonium chloride in an emulsion, its toxicity could be substantially reduced. A 0.1% CsA formulation (Cyclokat1) based on the Novasorb technology has recently been marketed in Europe for the treatment of DES under the brand name Ikervis1. In vivo studies comparing Ikervis with Restasis have shown a nearly two- to fourfold increase in corneal CsA concentrations after application of 0.05% and 0.1% Ikervis formulations, respectively [84]. Phase III clinical trials demonstrated a significant improvement in ocular inflammation and reduction in DES symptoms.

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However, adverse effects of the formulation were prominent, with nearly 30% of patients reporting pain and discomfort at the site of instillation [67]. A well-known approach to overcome the stability problems associated with nanoemulsions is by formulating solid lipid nanoparticles (SLNs), wherein the oil phase of o/w emulsions is replaced with a lipid that is solid at room temperature [143]. SLNs prepared using glyceryl behenate as the lipid matrix as well as poloxamer 188 and polysorbate 80 as the surfactants showed improved absorption and penetration of CsA across the cornea in vivo [144]. The improved bioavailability was hereby attributed to the small size of the SLNs, which could increase their accumulation in the conjunctival sac and, thus, improve precorneal retention. SLN retention could further be improved by the addition of a positive charge. Cationic chitosan-coated SLNs have shown improved accumulation in, and penetration across, the cornea compared with anionic or neutral SLNs [145]. The irritation potential of blank SLNs was found to be low ex vivo; however, further tests with epithelial cell lines as well as in vivo need to be performed to substantiate these results.

In situ gelling systems Over the past decade, in situ gelling systems have emerged as promising drug delivery systems because of their ease of administration, improved bioavailability, and ability to provide sustained release for a prolonged period of time [146]. Ophthalmic in situ gels comprise polymers that undergo reversible sol–gel transformation in response to a change in environmental and physiological factors, such as pH [147], ions [148], and temperature [149]. Ion-responsive ocular in situ gelling systems have been commonly prepared using divalent or trivalent polysaccharides, such as gellan gum and alginates [150]. The ions present in the tear fluid tend to interact which these gums to form 3D networks containing ionic interchange bridges, rapidly increasing the viscosity. Given that the osmolality of the tear fluid is consistently elevated in individuals with DES because of excessive evaporation of the tear fluid and/or inadequate fluid secretion [151–153], large ion concentrations are available for the gelling of ion-sensitive systems. Gan et al. [154] prepared CsA-loaded microemulsions by dispersing castor oil, Solutol HS 15 (surfactant), glycerol, and water in a gellan gum solution. A drastic increase in the viscosity of these systems was observed in tear fluid, resulting in increased precorneal residence time and, thus, higher CsA bioavailability with reduced toxicity. Thermosensitive gels prepared from poly-(N-isopropylacrylamide) (PNIPAAm), a well-known synthetic thermosensitive polymer that undergoes gel–sol transition at 32 8C, have also been suggested for ocular delivery of CsA. Wu et al. [155] conjugated PNIPAAm with natural biodegradable polymers, such as hyaluronic acid, to obtain thermosensitive and biodegradable microgels with high drug-loading capacities (Fig. 4). The hyaluronic acidgrafted PNIPAAm (HA-g-PNIPAAm) microgels were safe and nontoxic and significantly increased the precorneal residence time of CsA compared to conventional castor oil formulations and Restasis. Conjugates of HA and chitosan derivatives have also been used to prepare thermosensitive in situ nanogels for sustained delivery of CsA to the eye [156]. These nanogels had a high drug-loading capacity (up to 15.08%) and significantly increased

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Sonication

Incubation Drug loading T
T
T
HA main backbone

Drug release

HA-g-PNIPAAm polymers HA-g-PNIPAAm microgels

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Grafting PNIPAAm Cya Drug Discovery Today

FIGURE 4

Schematic representation of the HA-g-PNIPAAm microgel assembly and drug release process. HA-g-PNIPAAm microgels were prepared by sonication and cyclosporine A (CsA) was loaded by incubation. At temperatures below the lower critical solution temperature (LCST), hyaluronic acid-grafted PNIPAAm (HA-g-PNIPAAm) polymers exhibited good solubility in water; however, temperatures above the LCST affected the osmotic pressure of the HA-gPNIPAAm polymers, resulting in drug release [155].

CsA bioavailability, rendering them promising candidates for sustained CsA therapy. PLGA and PCL are also common polymers used in the preparation of in situ gelling systems because of their biodegradable and biocompatible properties. Thermosensitive diblock polymers have been prepared by coupling PLGA or PCL with methoxy-(polyethylene glycol) (mPEG) for ophthalmic delivery of CsA [157]. These diblock polymers underwent gel-to-sol transition by forming micelles at the physiological temperature of the eye and released CsA for up to 2 weeks in vitro. However, a significant limitation of such in situ gelling systems might be blurring of vision because of their increased viscosity, often resulting in higher patient discomfort and reduced adherence to therapy. Another approach to form in situ gels is by synthesizing lyotropic liquid crystalline systems, which are water-insoluble cubic phase dispersions of amphiphilic lipids that self-assemble in aqueous solutions to form highly viscous bicontinuous lyotropic crystals on the ocular surface [158,159]. Liquid crystalline nanoparticles based on glyceryl monooleate (GMO) and poloxamer 407 showed improved bioavailability and retention of CsA in the cornea, with good ocular tolerance in vitro [160]. However, further studies are required to establish the in vivo ocular tolerance and toxicity of such formulations. As discussed above, patients with DES have low moisture levels on the corneal surface because of extensive evaporation and/or insufficient secretion; thus, in situ gelling of the bicontinuous phase might be difficult to achieve in the clinical setting.

Hydrogels In recent years, contact lenses have emerged as a promising alternative for sustained ophthalmic drug delivery because of their ability to prolong corneal residence while being convenient and relatively easy to use [161]. The traditional method for preparing medicated hydrogel contact lenses is by soaking the lens in a drug solution. While soaked lens systems improve drug bioavailability slightly, they tend to release most of the drug within a few hours post administration and have minimal sustained-release effect [162,163]. Sustained release of drugs from lenses can be achieved by increasing their partition coefficient. Peng et al. [164] showed that 8

a silicon hydrogel lens could provide sustained release of CsA for up to 2 weeks in contrast to a soaked commercial lens, which could provide drug delivery for only a day. The diffusion barrier in a hydrogel can be further increased by incorporating the drug in sustained-release systems, such as nanoparticles within the hydrogel matrix, or by modifying the properties of the hydrogel matrix. For example, by incorporating vitamin E into the silicon hydrogel matrix, its viscosity and, therefore, the hydrogel diffusion barrier can be increased, prolonging the duration of drug release from 2 weeks to 1 month [165]. Collagen shields soaked in CsA liposome solutions have also been tested for sustained CsA release with improved precorneal residence time and tear film concentrations [166]. However, collagen shields are generally not well accepted because of their low transparency, which blurs the patient’s vision. In another study, the hydrogel diffusion barrier was increased by crosslinking poly-(2-hydroxyethyl methacrylate) (p-HEMA) with hydroxypropyl-b-cyclodextrin to achieve prolonged release of CsA over 3 months [167]. Cyclodextrin based p-HEMA hydrogels are able to solubilize both hydrophobic and hydrophilic drug molecules, making them extremely versatile. Kapoor et al. [168] found that p-HEMA hydrogels containing CsA microemulsions or Brij-97 micelles, respectively, could provide sustained CsA release for up to 20 days, whereas pure p-HEMA hydrogels could release the drug for up to 7 days only. Overall, the use of contact lenses and shields in patients with DES is rather limited because they tend to dry out the eye even more, resulting in increased discomfort and further aggravation of the condition. The discomfort is even more pronounced in the case of p-HEMA hydrogels lenses, because their poor oxygen permeability often leads to severe adverse effects upon prolonged use. To counter this limitation, Gupta et al. [169] fabricated a CsA-loaded p-HEMA core as a punctal plug by partially enclosing it in an impermeable membrane. It was predicted that this device would be able to release therapeutic concentrations of CsA for over 3 months. It was previously reported that unmedicated punctal plugs prevent tear drainage and, thus, increase the moisture levels in the eye. Thus, administering CsAloaded punctal plugs might have an additive effect in DES therapy [170], although the long-term use of punctal plugs might become uncomfortable and plugs have been shown to be expelled in several patients.

Concluding remarks Ocular drug delivery is a challenging prospect because of the penetration barriers presented by the cornea and conjunctiva. Although topical application remains the preferred method for drug delivery to the front of the eye, excipients need to be selected carefully to avoid toxicity to the highly sensitive ocular mucosa. This becomes even more challenging when formulating hydrophobic drugs, such as CsA, because most nonaqueous solvents are not generally recognized as safe (GRAS). Moreover, penetration enhancers and surfactants typically used to solubilize hydrophobic drugs in aqueous solutions often exhibit toxicity. Although several advanced drug delivery systems have been investigated and have shown potential for improving CsA delivery, further research is warranted to establish their long-term safety and tolerability profiles. This is especially the case when new excipients are used because they might have unknown long-term toxicity profiles and might not yet have sufficient safety data for regulatory approval. Another significant

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limitation of many new formulations is their complex formulation procedure and difficulty in manufacturing them at a commercial scale, rendering it expensive and impractical for such products to make the transition from bench to bedside. One of the most rational approaches for the development of CsA formulations has been the loading of the drug into simulated tear solutions already marketed for DES treatment. For example, Santen reformulated its innovative Novasorb technology (marketed as Retaine MGDTM in the USA and Canada), which is used as a tear substitute in mild DES, to develop Cyclokat for delivery of CsA to the eye. This product is currently marketed in Europe under the brand name Ikervis and Phase III clinical trials have shown a positive benefit:risk ratio for the treatment of severe DES [67]. A similar strategy was also used by Novaliq GmbH in the development of CyclASol, using the EyeSolTM artificial tear platform currently undergoing Phase II clinical trials. This novel SFA-based system does not require any surfactants or preservatives and Phase I trials showed excellent ocular tolerability [82]. CyclASol has a dual therapeutic effect because of the lubricating nature of the vehicle, while CsA reduces ocular inflammation and promotes long-term healing of the cornea. Finally, this formulation shows

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excellent stability and is easy to manufacture, making it a promising candidate for the treatment of DES in the future. DES is a widespread disease of the ocular surface, which causes significant discomfort in a large fraction of the population. Currently available CsA formulations have poor bioavailability and patient tolerability. Therefore, a safe and effective CsA dosage form is the need of the hour. Old age is one of the major predictors of DES, which highlights the importance of a safe, cost-effective, and easily accessible alternative that can conveniently reach the older population. Research and development of improved CsA formulations could help in significantly reducing the duration of treatment, frequency of drug administration, and discomfort resulting from adverse effects, thus improving the quality of life of patients with DES.

Conflict of interest The authors are currently consulting for Novaliq GmbH.

Acknowledgement The authors would like to thank Novaliq GmbH, Germany, for their financial support in form of a scholarship to P.A.

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97 Qi, L. et al. (2005) Cytotoxic activities of chitosan nanoparticles and copper-loaded nanoparticles. Bioorg. Med. Chem. Lett. 15, 1397–1399 98 Rupenthal, I.D. et al. (2011) Ion-activated in situ gelling systems for antisense oligodeoxynucleotide delivery to the ocular surface. Mol. Pharm. 8, 2282–2290 99 Aksungur, P. et al. (2011) Development and characterization of Cyclosporine A loaded nanoparticles for ocular drug delivery: cellular toxicity, uptake, and kinetic studies. J. Control. Release 151, 286–294 100 Pignatello, R. et al. (2002) Eudragit RS1001 nanosuspensions for the ophthalmic controlled delivery of ibuprofen. Eur. J. Pharm. Sci. 16, 53–61 101 Das, S. et al. (2010) Design of Eudragit RL 100 nanoparticles by nanoprecipitation method for ocular drug delivery. Nanomedicine 6, 318–323 102 Bucolo, C. et al. (2002) Enhanced ocular anti-inflammatory activity of ibuprofen carried by an Eudragit RS100. nanoparticle suspension. Ophthalmic Res. 34, 319–323 103 Zhao, X.L. et al. 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165 Peng, C.-C. et al. (2010) Extended delivery of hydrophilic drugs from siliconehydrogel contact lenses containing Vitamin E diffusion barriers. Biomaterials 31, 4032–4047 166 Pleyer, U. et al. (1994) Ocular absorption of cyclosporine a from liposomes incorporated into collagen shields. Curr. Eye Res. 13, 177–181 167 Bas¸bagˇ, A.B. et al. (2014) Poly(HEMA)/cyclodextrin-based hydrogels for subconjunctival delivery of cyclosporin A. J. Appl. Polym. Sci. Symp. 131 http:// dx.doi.org/10.1002/app.40397 168 Kapoor, Y. and Chauhan, A. (2008) Ophthalmic delivery of Cyclosporine A from Brij-97 microemulsion and surfactant-laden p-HEMA hydrogels. Int. J. Pharm. 361, 222–229 169 Gupta, C. and Chauhan, A. (2011) Ophthalmic delivery of cyclosporine A by punctal plugs. J. Control. Release 150, 70–76 170 Roberts, C.W. et al. (2007) Comparison of topical cyclosporine, punctal occlusion, and a combination for the treatment of dry eye. Cornea 26, 805–809 171 Kim, J. et al. (2010) Extended release of dexamethasone from silicone-hydrogel contact lenses containing vitamin E. J. Control. Release 148, 110–116

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