Development and Effects of FTY720 Ophthalmic Solution on Corneal Allograft Survival

Abstract

Fingolimod (FTY720), a novel class of sphingosine 1-phosphate receptor modulators, has received special interest among ophthalmologists, particularly given that oral administration of FTY720 has proven to effectively treat corneal graft rejection in animal models. However, no studies have examined the performance of FTY720 as an ophthalmic solution in reducing corneal rejection in high-risk corneal rejection models, and the stability and ocular irritation profile of FTY720 ophthalmic solution are also unknown. Thus, we developed 0.1%, 0.2% and 0.5% FTY720 ophthalmic solutions and evaluated their chemical stabilities under various storage conditions with high- performance liquid chromatography. To investigate the ocular irritancy of the FTY720 ophthalmic solution, New Zealand albino rabbits were subjected to the Draize test. Furthermore, classic, well-established rat allogenic penetrating keratoplasty models were used to investigate the anti-rejection efficacy of the tested FTY720 ophthalmic solutions. We found that the non-irritating 0.5% FTY720 ophthalmic solution could prolong corneal allograft survival in rats with significant efficacy for about one month. Furthermore, no significant concentration changes occurred in any of the types of FTY720 ophthalmic solutions within three months. These results revealed crucial profiles of FTY720 ophthalmic solutions and warrant further investigation and optimization of FTY720 in the anti-rejection therapy after keratoplasty.

Figure 1. Stability of FTY720 ophthalmic solutions under three storage conditions.

Condition 1: 25 °C with protection from light; Condition 2: 25 °C without protection from light; Condition 3: 40 °C with protection from light. (a) 0.1% FTY720 ophthalmic solution; (b) 0.1% FTY720 ophthalmic solution; (c) 0.5% FTY720 ophthalmic solution.

Figure 2. Ocular irritant scores for the FTY720 ophthalmic solutions at the end of the Draize test.

Topical reactions were observed under a slit-lamp microscope after the in vivo instillation of normal saline or the 0.1% FTY720, 0.2% FTY720 or 0.5% FTY720 ophthalmic solution for 7 days. (a) Normal saline (control group); (b–d) Topical instillation of 0.1%, 0.2% or 0.5% FTY720 ophthalmic solutions, respectively.

Figure 3. Clinical manifestations and histopathology (HE staining, magnification, 400×) of corneal grafts on postoperative day 14.

(a) Control group; (b) Systemic FTY720 treatment group (1.2 mg/kg/d); (c) Topical instillation of 0.05% FK506 ophthalmic suspension group; (d-f) Topical instillation of 0.1%, 0.2% or 0.5% FTY720 ophthalmic solution groups, respectively. In (a,d,e), the pupil and iris vessels are not visible due to the severe opacity and edema of the grafts. Moreover, the degree of inflammatory cell infiltration was much greater in (a,d,e) than in (b,c,f), consistent with the clinical manifestations. S, suture knots; G, corneal grafts (the area surrounded by eight suture knots); P, pupil; EPI, epithelial layer; END, endothelial layer.

Figure 4. Survival curves of corneal allografts for all groups.

Treatment with 0.5% FTY720, 0.05% FK506 and systemic FTY720 (1.2 mg/kg/d) significantly prolonged graft survival compared with the control condition (all p < 0.01; n = 8) or treatment with 0.1% FTY720 or 0.2% FTY720 (all p < 0.01; n = 8). Allograft survival of the control group was not significantly different from that in the 0.1% FTY720 ophthalmic solution treatment group (p = 0.08, n = 8) but was significantly different from that of the 0.2% FTY720 treatment group (p < 0.01; n = 8). Systemic treatment with FTY720 and topical treatment with 0.05% FK506 resulted in longer allograft survival than treatment with the 0.5% FTY720 ophthalmic solution (p = 0.03 and 0.01, respectively). No significant differences were observed between the results obtained after treatment with systemic FTY720 and those obtained after treatment with topical 0.05% FK506 (p = 0.59; n = 8).

Figure 5. Average clinical finding scores of corneal rejection for all groups throughout the entire follow-up period.

(a) Mean rejection scores; (b) Mean opacity scores; (c) Mean edema scores; (d): Mean neovascularization scores. The results obtained for the 0.5% FTY720 group, the oral FTY720 group and the 0.05% FK506 group were significantly lower than those obtained for the control group (all p < 0.05).