Tomoko Oda

Cyclosporin A eye drops inhibit fibrosis and inflammatory cell infiltration in murine type I allergic conjunctivitis without affecting the early-phase reaction.

Purpose: To understand the mechanisms of action of cyclosporin A eye drops in severe allergic diseases such as vernal keratoconjunctivitis, the inhibitory effects of cyclosporin A eye drops on fibrosis and inflammatory cell infiltration in murine allergic conjunctivitis were evaluated. Methods: BALB/c mice that had been actively sensitized with ovalbumin were challenged with ovalbumin on days 10–14 after initial sensitization. Cyclosporin A (0.1%) or betamethasone (0.1%) eye drops were instilled 1, 4, and 7 hours after each challenge. Ocular tissue was harvested for histological evaluation 24 hours after the last challenge, and conjunctival tissue was collected for the measurement of collagen content and quantitative PCR 8 hours after the last challenge. Results: Scores for fibrosis and inflammatory cell infiltration and collagen content in the conjunctiva were higher after 5 days of antigen challenge than in normal non-challenged conjunctiva. Instillation of cyclosporin A or betamethasone reduced the antigen-induced increases in scores for fibrosis and inflammatory cell infiltration in the conjunctiva, and cyclosporin A significantly reduced the antigen-induced increase in conjunctival collagen content. Betamethasone also showed a tendency to reduce the increase in collagen content. Cyclosporin A and betamethasone decreased the numbers of CD3+ and CD4+ T-cells and eosinophils in the conjunctiva, but did not affect the number of mast cells. Neither type of eye drop suppressed the increase in vascular permeability that occurred for 30 minutes after the last antigen challenge. In quantitative PCR, cyclosporin A suppressed the expression of IL-4 and IL-5 mRNA but did not suppress the expression of transforming growth factor (TGF)-β 1, whereas betamethasone suppressed the expression of IL-4, IL-5, and TGF-β 1. Conclusion: The results suggest that cyclosporin A eye drops inhibited fibrosis and inflammatory cell infiltration by the suppression of Th2 cytokine production in repeatedly antigen-challenged conjunctiva without affecting the early-phase reaction.

Beneficial pharmacological effects of selective glucocorticoid receptor agonist in external eye diseases.

PURPOSE Glucocorticoids exert their actions via the glucocorticoid receptor through at least 2 intracellular mechanisms, known as transrepression and transactivation. It has been hypothesized that transrepression is the basis of their anti-inflammatory effects, whereas transactivation has been assumed to cause their side effects. ZK209614, a recently identified, novel selective glucocorticoid receptor agonist, exerts strong transrepression and weak transactivation. The objective of this study was to determine whether its pharmacological effects can be dissociated from its side effects. For this, we employed in vitro assays and topical in vivo models. METHODS ZK209614 and dexamethasone were used in in vitro transrepression and transactivation assays. To evaluate anti-inflammatory and antiallergic activities in vivo, ZK209614 and betamethasone phosphate were tested in the carrageenan-induced conjunctivitis model and allergic conjunctivitis model in rats. To evaluate side effects in vivo, treatments with ZK209614 and betamethasone phosphate were tested for the ocular hypertensive effects in a feline model, each drug being administered topically. RESULTS ZK209614 showed strong transrepression and weak transactivation in the in vitro assays. When given as eyedrops, ZK209614 and betamethasone phosphate each had an inhibitory effect on edema weight in the rat carrageenan-induced conjunctivitis model. In the rat allergic conjunctivitis model, ZK209614 reduced the elevated vascular permeability at a concentration of 0.1%. In the feline intraocular pressure (IOP)-elevation experiment, topically administered betamethasone phosphate elevated IOP, but ZK209614 had no effect on IOP. CONCLUSION The present investigations suggest that ZK209614 eyedrops have both anti-inflammatory and antiallergic effects, but no unwanted IOP-elevating effect. On that basis, ZK209614 might be a promising candidate as an ophthalmic drug with a better therapeutic index than classic glucocorticoids.

The role of platelet activating factor and the efficacy of apafant ophthalmic solution in experimental allergic conjunctivitis.

Platelet-activating factor (PAF) may be an important mediator in allergic conjunctivitis. In this study, apafant, a potent PAF antagonist, was evaluated for topical ocular anti-PAF activity in PAF and antigen stimulated conjunctivitis models. PAF, when injected into parpebral conjunctiva, provoked an acute increase, measured as dye leakage, in conjunctival vascular permeability. Apafant inhibited this response in a dose-related manner, and the inhibitory action of 0.1% apafant lasted for at least 6 hours duration. PAF, when instilled into the conjunctival sac, induced itch-scratching behavior and clinical symptoms, such as conjunctival redness and edema. These were inhibited by pretreatment with apafant ophthalmic solution. In a passive conjunctival anaphylaxis model in guinea pigs, significant inhibition of the allergic response was observed following topical ocular administration of apafant 5 and 15 minutes prior to the antigen challenge. We have demonstrated that PAF plays an important role in the development of allergic conjunctivitis. These results clearly indicate that apafant has potential as a topical ocular anti-PAF for treating allergic conjunctivitis.

Meibomian Gland Dysfunction Model in Hairless Mice Fed a Special Diet With Limited Lipid Content.

PURPOSE A novel meibomian gland dysfunction (MGD) model was developed to facilitate understanding of the pathophysiology of MGD and to evaluate treatment with azithromycin ophthalmic solution (azithromycin). MGD was induced in HR-1 hairless mice by feeding them a special diet with limited lipid content (HR-AD). METHODS Male HR-1 hairless mice were fed an HR-AD diet for 16 weeks. Development of MGD was assessed by histopathology at 4-week intervals. The lid margin was observed by slit-lamp examination. After cessation of the HR-AD diet, the mice were fed a normal diet to restore normal eye conditions. Expression of cytokeratin 6 was determined by immunostaining. We evaluated the effects of topically applied azithromycin on the plugged orifice in this model. RESULTS After mice were fed the HR-AD diet, histopathology analysis showed hyperkeratinization of the ductal epithelium in the meibomian gland. Ductal hyperkeratinization resulted in the loss of acini, followed by atrophy of the gland. Slit-lamp examination revealed a markedly plugged orifice, telangiectasia, and a toothpaste-like meibum compared with that of a normal eyelid. Cessation of feeding with HR-AD ameliorated both the MGD signs and the expression of cytokeratin 6, restoring the tissue to a histologically normal state. Azithromycin treatment significantly decreased the number of plugged orifices and ameliorated atrophy, as revealed by histopathologic analysis. CONCLUSIONS We developed a novel model that mimics human MGD signs in HR-1 hairless mice fed an HR-AD diet. Azithromycin treatment led to therapeutic improvement in this model. This MGD model could be useful for the evaluation of drug candidates for MGD.

Cyclosporine A Eye Drops Inhibit the Early-Phase Reaction in a Type-I Allergic Conjunctivitis Model in Mice

PURPOSE The effects of cyclosporine A eye drops on the early-phase reaction were investigated in a type-I allergic conjunctivitis model. METHODS Mice were actively sensitized with ragweed (RW) absorbed on aluminium hydroxide gel and challenged with RW for 10 days (single challenge model) or 10-14 days (repetitive challenge model) after the first sensitization. For the evaluation of itching, ovalbumin was used as an antigen instead of RW. The effects of cyclosporine A eye drops on increased vascular permeability, mast cell degranulation, and itching were evaluated and compared with those of other anti-allergic eye drops. RESULTS In the single challenge model, cyclosporine A eye drops significantly inhibited the increase in vascular permeability and histological evaluations showed suppressed degranulation of mast cells. Disodium cromoglycate (DSCG) eye drops showed only a slight tendency to inhibit the increase in both pathophysiological parameters. Ketotifen or betamethasone eye drops significantly inhibited the increase in vascular permeability. The order of potency in the single challenge model was ketotifen > cyclosporine A > betamethasone. In the repetitive challenge model, cyclosporine A eye drops significantly inhibited the increase in vascular permeability and DSCG eye drops showed only slight inhibition. Ketotifen or betamethasone significantly inhibited the increase in vascular permeability. The order of potency in the repetitive challenge model was cyclosporine A > betamethasone > ketotifen. The effect of cyclosporine A eye drops on the itch-scratch response was studied. Cyclosporine A and DSCG significantly reduced the itch-scratch response in the single and repetitive challenge models; the effect of cyclosporine A in the repetitive challenge model was more potent than in the single challenge model. CONCLUSIONS Those results suggest that administration of cyclosporine A eye drops inhibit the early-phase reaction in type-I allergic conjunctivitis, which may be mediated by the suppression of mast cell degranulation. This action of cyclosporine A eye drops may be involved in the therapeutic effect of cyclosporine A on allergic conjunctivitis.

Transfer of a T-helper type 2 clone into conjunctiva induces corneal damage in mice.

Purpose: We established a T-helper Type 2 (Th2) clone-induced conjunctival eosinophilia model by injecting D10.G.4.1 (D10) cells, a murine Th2 clone, and conalbumin, its specific antigen, into conjunctiva of AKR/J mice. Using this model, we investigated the effect of a coinjection of D10 cells and conalbumin into conjunctiva on corneal damage. Methods: Corneal fluorescein staining scores and eosinophil peroxidase (EPO) activity in conjunctiva were measured after coinjection of D10 and conalbumin into conjunctiva, and the effects of cyclosporine A, betamethasone, and anti-interleukin-5 antibody on staining scores and EPO activity were examined. Results: Coinjection of D10 and conalbumin induced an increase of the corneal fluorescein staining score after 24, 48, and 96 hours and 10 days. EPO activity in conjunctiva increased time-dependently until 24 hours after coinjection. The increase in the staining score followed the time dependent increase in EPO activity. The instillation of cyclosporine A, an inhibitor of cytokine production from T-cells, and betamethasone significantly inhibited the increase in corneal fluorescein score and EPO activity. Intraperitoneal administration of anti-interleukin-5 monoclonal antibody, which inhibits the infiltration of eosinophils into the conjunctiva, completely inhibited the increase in staining score. Conclusion: The transfer of the Th2 clone into the murine conjunctiva induced corneal damage, which may have been caused by Th2 cell-produced interleukin-5 that mediated the activation of eosinophils.

A Novel Model of Meibomian Gland Dysfunction Induced with Complete Freund’s Adjuvant in Rabbits

A novel meibomian gland dysfunction (MGD) model induced by the injection of complete Freund’s adjuvant (CFA) in rabbits was developed to facilitate the understanding of the pathophysiology of MGD with meibomitis. In addition, we sought to evaluate treatment with steroid eye drops in this model. Male Japanese white rabbits were subcutaneously injected with CFA into the upper eyelid margin. The eyelid margins of the rabbits were chronologically observed through slit lamp examination. The development of meibomitis was assessed through histopathology. We evaluated the effects of topically applied tobramycin/dexamethasone (Tob/Dex) eye drops on the plugged orifices and telangiectasia. After the injection of CFA, slit lamp examination revealed markedly plugged orifices, telangiectasia around the orifices and a toothpaste-like meibum, as compared with the normal eyelids. Histopathology revealed granulation tissue with infiltration of inflammatory cells, hyperkeratinization of the ductal epithelium, and cystic dilatation of ducts in the meibomian gland. The orifices were plugged with a proteinaceous substance. Tob/Dex eye drops significantly suppressed the plugging and telangiectasia around the orifices. Through the injection of CFA, we successfully established a novel rabbit MGD that mimics the symptoms observed in humans meibomitis. This model should be useful in the evaluation of the efficacy of drug candidates.