Ординатура / Офтальмология / Английские материалы / Ocular Therapeutics Eye on New Discoveries_Yorio, Clark, Wax_2007

mediators in ocular tissue were reduced by treatment of dry eye patients with cyclosporine 0.05%. Statistical reductions of CD40 (P 0.049) and CD40 ligand (P 0.008), and in the percentage of cells expressing Fas (P 0.046) were identified as evidence of suppression of apoptosis (Brignole et al., 2000).

The pharmacology of cyclosporine is very interesting and many obstacles to its clinical use in dry eye were overcome in bringing it to market. Cyclosporine A is a cyclic undecapeptide that is a product of the fungi Tolypocladium inflatum and Beauveria nevus. It is lipophilic and not easily dissolved in aqueous solution. Initially the mechanism of action was thought primarily to inhibit activation of T-lymphocytes, but further study has verified the molecule’s multiple inhibitory properties, including the ability to inhibit apoptosis in other cell types. The cyclosporine A molecule binds to two cytoplasmic proteins, cyclophilin A and cyclophilin D (Figure 6.3). The cyclosporine A–cyclophilin A complex inhibits calcineurin activity resulting in inhibition of T-lymphocyte activation. Interference with the nuclear factor for T-cell activation (NFAT) results in reduced intracellular calcium induction by antigen binding to the T-cell which abrogates activation of the lymphocyte (Crabtree and Olson, 2002).

The inhibition of apotosis (programmed cell death) by cyclosporine appears to occur as a result of binding cyclosporine A to cyclophilin D (Waldmeier et al., 2003). The cyclosporine A–cyclophilin D complex interferes with initiation of the apoptosis cascade which otherwise results in cellular stress or damage. In an experimental murine model of dry eye, cyclosporine A significantly reduced apoptosis of conjunctival epithelial cells, as measured by DNA fragmentation and activated caspase-3 levels (Strong et al., 2005).

Preparation of cyclosporine for topical ocular use was a challenge due to its hydrophobicity. Cyclosporine A was initially prepared for topical ocular use in oil or ointment vehicles which were messy and uncomfortable to use, and the concentration and tissue partitioning properties were limited in these vehicles. A major advance in clinical application of cyclosporine A was the development of a lipid emulsion formulation in castor oil that also included glycerin, polysorbate 80, and sodium hydroxide (to adjust the pH) that was better tolerated and allowed higher concentration of cyclosporine delivery to the eye. Animal studies confirm that topical administration of the emulsion results in sufficient cyclosporine concentrations to achieve immunomodulation in

both cornea and conjunctiva, but with very low concentrations ( 1 ng/mL) in aqueous humor, vitreous humor and plasma (Small et al., 2002). Topical administration of cyclosporine 0.05% or 0.1% ophthalmic emulsions in human subjects achieves plasma levels of cyclosporine that are undetectable in those patients receiving 0.05% cyclosporine, and are very low in those receiving 0.1% (Sall et al., 2000). This level of concentration is several orders of magnitude lower than trough plasma concentrations of cyclosporine achieved during systemic immunosuppressive therapy for psoriasis, rheumatoid arthritis, and organ transplant patients (75 to 400 ng/mL) (Tang-Liu and Acheampong, 2005). The drug has proved to be very safe and systemic side effects were not observed in clinical trials (Sall et al., 2000; Stevenson et al., 2000; Barber et al., 2005). The adverse effects are listed in Table 6.1 and include stinging upon instillation, but without evidence of superinfection or toxicity.

The clinical response to topically applied cyclosporine 0.05% emulsion is beneficial in many patients. Phase II clinical trials were conducted in moderate to severe dry eye patients (Schirmer 5 mm/5 min; corneal fluorescein staining 4/15; symptoms 2/4) with twice daily dosing. The randomized, double-masked, placebo-controlled, doseranging clinical trial enrolled 90 patients with moderate to severe dry eye disease for a

12 week treatment (Stevenson et al., 2000). Cyclosporine 0.05% and 0.1% emulsions produced significant improvements from baseline symptoms, corneal fluorescein, and rose bengal staining, but no clear dose response relationship was observed. Two expanded Phase III clinical trials were subsequently conducted in a similar patient population and determined the efficacy of cyclosporine 0.05% and 0.1% ophthalmic emulsions. The two identical randomized, double-masked, placebo-controlled trials analyzed 877 moderate to severe dry eye patients who were randomly treated with instilled cyclosporine 0.05% (n 293), cyclosporine 0.1% (n 292), or the emulsion vehicle (n 292) BID for 6 months (Sall et al., 2000). Cyclosporine 0.05% provided the most consistent improvement, but the emulsion vehicle was also very effective in reducing some signs and symptoms, particularly in the early follow-up period. This prominent vehicle effect prevented demonstration of statistically significant difference between vehicle and active drug, but the prominent response to the vehicle prompted subsequent marketing of the vehicle as a tear-stabilizing lubricant as previously discussed as enhanced tear supplement therapy (Di Pascuale et al., 2004)

Cyclosporine 0.05% emulsion therapy produced significantly greater improvement in blurred vision than vehicle-treated patients after 1, 3, 4, and 6 months (P 0.014).

This group of patients was able to decrease the frequency of supplemental artificial tear use compared with those in the vehicle group after 6 months of therapy (P 0.006). Significantly better reduction in punctate corneal fluorescein staining than vehicle occurred with cyclosporine 0.05% at 4 and 6 months of treatment (P 0.044 and P 0.008, respectively). Increase in tear production as assessed by anesthetized Schirmer testing was significantly greater in the cyclosporine 0.05% group than in the vehicle group (P 0.009). Burning/stinging (at 4 and 6 months) and itching (at 1, 3, 4, and 6 months) decreased from baseline in the cyclosporine 0.05% group (P 0.024 and P 0.002, respectively) and were consistent with improvement in clinical signs, but the symptomatic improvement was not significantly different from that occurring with the vehicle.

Approval of 0.05% cyclosporine ophthalmic emulsion by the Food and Drug Administration (FDA) was obtained after subsequent subset analysis of the beneficial effect of cyclosporine on tear production in those patients who were not concomitantly treated with other anti-inflammatory therapy or previously treated with punctal plugging. The approval was for the indication of reduced tear production presumed to be due to inflammation. Improvement in tear production was seen in 59% of patients, with 15% of patients demonstrating

10 mm or more increase in Schirmer testing (Figure 6.4).

In addition to the improved clinical outcome, immunohistological improvement of the ocular surface abnormalities occurred with topical cyclosporine therapy, including reduction of cell surface markers of activated T-lymphocytes and apoptotic cells in conjunctival biopsies (Figures 6.1 and 6.2). Cyclosporine treatment reduced expression of pro-inflammatory cytokine as well. Cyclosporine 0.05% was significantly more effective in reducing HLA-DR levels than treatment with vehicle (P 0.034) (Strong et al., 2005). A second study confirmed significant reductions of HLA-DR and a marker of activated T-cells, CD11a, in conjunctival biopsies from dry eye patients following 6 months of cyclosporine 0.05% treatment (P 0.05) (Sall et al., 2000; Kunert et al., 2000). Pro-inflammatory cytokine levels in the tears and/or conjunctival epithelium of patients with dry eye were reduced following therapy with topical cyclosporine A emulsion (Brignole et al., 2000). Levels of IL-6 mRNA in conjunctival epithelial biopsy samples from dry eye patients treated with cyclosporine 0.05% for 6 months showed a significant decrease in IL-6 mRNA relative to pretreatment biopsies when analyzed by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) (Turner et al., 2000).

In addition to the markers of inflammation, cyclosporine has been shown to improve apoptosis. The molecular markers of apoptosis in conjunctival epithelia including CD40, CD40 ligand (CD40L, also known as CD154), and Fas decrease after topical cyclosporine 0.05% ophthalmic emulsion. Statistical reductions of CD40 (P 0.049) and CD40 ligand (P 0.008), and in the percentage of cells expressing Fas (P 0.046) were observed (Brignole et al., 2000). Such results are consistent with rodent studies that demonstrate reductions in the number of apoptoic epithelial cells and in levels of an activated protease that is important in apoptosis (caspase 3) following cyclosporine treatment (Gao et al., 1998b).

The improvement of health of the ocular surface epithelium following cyclosporine topical therapy has also been demonstrated by restoration of the goblet cell density in conjunctival epithelium. The goblet cells of the conjunctiva are a sensitive marker of damage from ocular surface disease and are decreased in chronic dry eye (Nelson, 1988). Since these cells provide the important gelforming MUC5AC which helps maintain the health of the ocular surface epithelium by protecting the surface cells, loss of goblet cells disturbs the homeostasis of the ocular surface (Zhao et al., 2001). Conjunctival goblet cell density increased following cyclosporine therapy by 191% (P 0.014), which was significantly greater than in biopsies of vehicle-treated patients (P 0.013) (Sall et al., 2000; Kunert et al., 2002).

The clinical results and the immunohistological response in dry eye patients indicate that cyclosporine 0.05% ophthalmic emulsion is the first medication available that, rather than only alleviating symptoms, actually treats an underlying cause of dry eye disease.

C. Essential Fatty Acids (Omega 3)

Another avenue of therapy thought to be due to suppression of inflammation is the systemic use of oral omega 3 essential

fatty acids (Horrobin, 1986). These fatty acids are required in dietary supplementation since the body does not produce such substances, and the general diet in the USA includes more omega 6 than omega 3 essential fatty acids. Epidemiologic studies have correlated an enhanced diet of tuna fish, which is high in omega 3 fatty acids, with a lower risk of chronic dry eye (Miljanovic, 2005). Recommendations for diet supplementation with sources rich in omega 3 fatty acids, such as fish oil or flax seed oil, have been made based upon such preliminary data and the findings of two clinical trials documenting improvement in dry eye symptoms and signs. One such study was conducted in patients with chronic dry eye disease and the other in patients following refractive surgery (Barabino et al., 2003; Macri et al., 2003). Multiple over-the-counter formulations for omega 3 fatty acids are available and one formulation (Theratears Nutrition™, Advanced Research, Inc.) is marketed specifically for dry eye therapy.

D. Secretagogues

It has long been a goal in the treatment of dry eye disease to identify therapeutic agents that could stimulate tear production. Such drugs are considered secretagogues. There are presently no secretagogues approved for stimulating tear production, but two of the systemically administered drugs that are approved for treating dry mouth by stimulating salivation, do have a stimulatory effect on the lacrimal glands. These drugs are indicated for the treatment of Sjögren’s syndrome related xerostomia, but clinical trials have also shown stimulation of lacrimation (Nelson et al., 1998; Mathers and Dolney, 2000). Pilocarpine (Salagen™, MGI-Pharma) was the first secretagogue clinically available for use. A prospective, randomized clinical trial verified efficacy in improving symptoms of dry mouth with a smaller effect on symptoms of dry eye (Vivino et al., 1999; Vivino, 2001). Cevimeline (Evoxac™, Daiichi

Mean Corneal Stain

Mean corneal staining (Average of all areas; 0–3 Scale)

1.5

Placebo

1.4

2% diquafosol

1.3

Off Treatment

1.1

1.0

0.9

0.8

0.7

Study Week

Pharmaceutical Company) is also approved in the USA for treatment of symptoms of dry mouth and, although not approved for the symptom of dry eye, it is better tolerated than pilocarpine and appears effective in both xerostomia and keratoconjunctivitis sicca at the 30 mg dose (Petrone et al., 2002). Side effects of sweating and diarrhea often limit the tolerance to the medication.

Considering the side effect limitations to the use of systemic secretagogues, topical secretagogues have been pursued with some success. Although not yet FDA approved for clinical use, the Phase II and Phase III clinical trials of a promising medication to increase aqueous tear volume and stimulate mucin secretion suggest that a novel P2Y2 receptor agonist (diquafosol, Inspire Pharmaceuticals, Inc.) is safe and effective in treating dry eye. This topical agent has been shown to increase the flow of sodium and water across conjunctival membranes and to stimulate mucin production from goblet cells (Li et al., 2001). Preliminary clinical trials demonstrate an amelioration of clinical symptoms and improvement of ocular

surface staining in dry eye patients with clearingofthecentralcornealstaining(Foulks et al., 2001; Tauber et al., 2004) (Figure 6.5). Other topical secretagogues including rebamipide (Otsuka/Novartis, Inc.) which appears to stimulate mucin production and improves symptoms and surface staining in dry eye, ecabet (Ista Pharmaceuticals, Inc.), and Moli1901 (Lantibio, Inc.) are under evaluation in Phase II clinical trials and are expected to proceed to Phase III trials in the very near future.

The early reports of preliminary results in the Phase II trial indicate that rebamipide reduces ocular surface staining and is well tolerated (Donshik PC and associates, Association for Research in Vision and Ophthalmology annual meeting, 2006).

E. Autologous Serum

The topical application of autologous serum has been found to be beneficial in treating patients with severe dry eye when other treatments have failed (Fox et al., 1984; Tsubota et al., 1999). It is thought that the

serum contains proteins, peptides and nutrients, as well as growth factors that protect and heal the ocular surface. The use of this therapy has been best described by Kasuo Tsubota and associates, who have reported a straightforward method of preparation and good results in therapy (Tsubota and Higuchi, 2000). Although the method is cumbersome, and associated with some risk of infection if contamination of the serum drops occurs, the results have been dramatic in some cases of dry eye, as well as persistent epithelial defects of the cornea (Noble et al., 2004). Identification of the specific molecules responsible for the healing effect of serum may allow more specifically targeted therapy in the future treatment of dry eye disease. The treatment technique may have wider application than just dry eye and persistent epithelial defect (Plugfelder, 2006).

F. Hormone Therapy

BOX 6.1

Hormonal support therapy is the most recent area of investigation in clinical treatment of dry eye. The strong laboratory evidence associating decreased androgen levels with lacrimal gland inflammation and lacrimal insufficiency suggest that androgen supplementation is a reasonable therapeutic option for dry eye disease (Sullivan et al., 1999; Sullivan, 2004). Additionally, epidemiologic evidence suggests that systemic estrogen-only supplementation not only improves symptoms of dry eye, but actually aggravates symptoms of ocular irritation (Schaumberg et al., 2001). There is, however, accumulating evidence that topical estrogen may be a viable treatment for dry eye due to a salutary effect on ocular surface epithelial cells (iDestrin estradiol, Nascent Pharmaceuticals, Inc.). Systemic therapy with a combination of estrogen and androgen (Estratest) has been documented

to improve symptoms of dry eye in a small retrospective study (Scott et al., 2005). Clinical trials evaluating topical testosterone are in Phase II trials but anecdotal studies with testosterone applied topically to the eyelids have claimed improvement in dry eye symptoms without associated complications (O’Connor, presentation at Association for Research in Vision and Ophthalmology, 2006).

V. CONCLUSION

Dry eye disease is a multifactorial disease that is aggravated by environmental conditions and activities to which the patient is exposed. Occurrence as an episodic condition can be controlled by palliative therapy provided by many OTC products, but the occurrence of chronic dry eye requires enhanced tear stabilization and efforts to protect the ocular surface from dessication and hyperosmolar stress. Additional treatment with anti-inflammatory therapy is necessary to control the inflammatory aspects of the disease. Therapy with secretagogues offers additional beneficial effect to protect the ocular surface. Hormonal supplementation and restoration of an appropriate balance between estrogen and androgen effects on the lacrimal system and the ocular surface may provide even better future management of chronic dry eye disease. The more we learn about the pathophysiology of dry eye disease, the better will our therapy become.

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I. CLINICAL DISEASE

Surface ablation photorefractive keratectomy (PRK) was the original procedure utilized to correct refractive errors using the excimer laser. Beginning in approximately 1988, clinical trials led to the application of PRK to correct low myopia, followed over a period of years by myopic astigmatism, high myopic astigmatism, hyperopia, hyperopic astigmatism, and mixed astigmatism. Around 1996, laser-assisted in situ keratomileusis (LASIK) was introduced to facilitate correction of these same refractive errors and this procedure, involving the formation of an epithelial–stromal flap, quickly became the dominant procedure in refractive surgery (Wilson, 2004). There are several reasons why LASIK became dominant over PRK, including faster visual

recovery and improved patient comfort, but a major factor that led to PRK surface ablation’s fall from dominance was the development of anterior stromal opacity (haze) in a proportion of patients who underwent the procedure (Figure 7.1).

Haze occurs, at least to a limited extent, in most human eyes that undergo excimer laser surface ablation (Wilson, 2004). This includes not only PRK, but also subsequent surface ablation procedures developed to overcome some of the limitations of PRK, including laser epithelial keratomileusis (LASEK), in which a solution of ethanol is used to facilitate formation of an epithelial flap, and Epi-LASIK, in which a microkeratome is used to generate an epithelial flap. In most corneas that undergo PRK, LASEK, or Epi-LASIK, haze formation is mild and does not significantly affect the